Pegylated OXM variants

ABSTRACT

A composition which includes oxyntomodulin and polyethylene glycol polymer (PEG polymer) linked via a reversible linker such as 9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl (FMS) is disclosed. Pharmaceutical compositions comprising the reverse pegylated oxyntomodulin and methods of using same are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.14/405,597, filed Dec. 4, 2014 and of U.S. application Ser. No.14/592,641, filed Jan. 8, 2015, which are both continuation in part ofPCT International Application No. PCT/IL2013/050481, InternationalFiling Date Jun. 4, 2013, claiming priority to U.S. Provisional PatentApplication No. 61/655,367, filed Jun. 4, 2012, all of which areincorporated by reference herein in their entirety. This application isalso a continuation in part of U.S. application Ser. No. 14/123,106,filed Nov. 28, 2013, which is a National Phase Application of PCTInternational Application No. PCT/US2012/040744, filed Jun. 4, 2012,which claims priority from U.S. Provisional Application Ser. No.61/492,448, filed Jun. 2, 2011, and U.S. Provisional Application Ser.No. 61/624,589, filed Apr. 16, 2012.

FIELD OF INVENTION

A composition which includes oxyntomodulin and polyethylene glycolpolymer (PEG polymer) linked via a reversible linker such as9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) is disclosed. Pharmaceutical compositions comprising the reversepegylated oxyntomodulin and methods of using same are also disclosed.

BACKGROUND OF THE INVENTION

The gastrointestinal tract is responsible on synthesize and releasing ofmany peptide hormones that regulate eating behavior including pancreaticprotein (PP), glucagon-like peptide 1 (GLP-1), peptide YY (PYY) andOxyntomodulin (OXM). OXM arises from a tissue-specific post-transitionalprocessing of proglucagon in the intestine and the CNS. It contains 37amino acids, including the complete glucagon sequence with a C-terminalbasic octapeptide extension that was shown to contribute to theproperties of OXM both in-vitro and in-vivo but was not alone sufficientfor the effects of the peptide. In response to food ingestion, OXM issecreted by intestinal L cells into the bloodstream proportionally tothe meal caloric content.

OXM enhances glucose clearance via stimulation of insulin secretionafter both oral and intraperitoneal administration. It also regulatesthe control of food intake. Intracerebroventricular (ICV) andintranuclear injection of OXM into the paraventricular and arcuatenuclei (ARC) of the hypothalamus inhibits re-feeding in fasting rats.This inhibition has also been demonstrated in freely fed rats at thestart of the dark phase. Moreover, peripheral administration of OXMdose-dependently inhibited both fast-induced and dark-phase food intake.

Unfavorable pharmacokinetics, such as a short serum half-life, canprevent the pharmaceutical development of many otherwise promising drugcandidates. Serum half-life is an empirical characteristic of amolecule, and must be determined experimentally for each new potentialdrug. For example, with lower molecular weight protein drugs,physiological clearance mechanisms such as renal filtration can make themaintenance of therapeutic levels of a drug unfeasible because of costor frequency of the required dosing regimen.

Proteins and especially short peptides are susceptible to denaturationor enzymatic degradation in the blood, liver or kidney. Accordingly,proteins typically have short circulatory half-lives of several hours.Because of their low stability, peptide drugs are usually delivered in asustained frequency so as to maintain an effective plasma concentrationof the active peptide. Moreover, since peptide drugs are usuallyadministered by infusion, frequent injection of peptide drugs causeconsiderable discomfort to a subject. Thus, there is a need fortechnologies that will prolong the half-lives of therapeutic proteinsand peptides while maintaining a high pharmacological efficacy thereof.Such desired peptide drugs should also meet the requirements of enhancedserum stability, high activity and a low probability of inducing anundesired immune response when injected into a subject.

The present invention relates to OXM derivative in which the half-lifeof the peptide is prolonged utilizing a reversible pegylationtechnology.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a composition consisting ofan oxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein said PEG polymer is attached to the amino terminus ofsaid oxyntomodulin via Fmoc or FMS.

In one embodiment, the invention relates to a composition consisting ofan oxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein said PEG polymer is attached to a lysine residue onposition number twelve (Lys₁₂) of said oxyntomodulin's amino acidsequence via Fmoc or FMS.

In another embodiment, the invention relates to a composition consistingof an oxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein said PEG polymer is attached to a lysine residue onposition number thirty (Lys₃₀) of said oxyntomodulin's amino acidsequence via Fmoc or FMS.

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 12 or to the Lysine residue on positionnumber 30 or to the amino terminus of said oxyntomodulin's amino acidsequence via 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the invention relates to a method of reducing thedosing frequency of oxyntomodulin, consisting of the step of conjugatinga polyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 12 or to the Lysine residue on position number 30 or tothe amino terminus of said oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to a Lysine residue on positionnumber 12 or to a Lysine residue on position number 30 or to the aminoterminus of said oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, the inventions of which can be better understood byreference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A and 1B show different variants of the PEG-FMS-OXM conjugateproduced (FIG. 1A: homogeneous product; FIG. 1B: heterogeneous product).

FIG. 2 is a graph showing the in vitro activity (cAMP quantitation) ofthe heterogeneous PEG₃₀-FMS-OXM and the three PEG₃₀-FMS-OXM variants(amino, Lys12 and Lys30) when incubated with CHO-K1 cellsover-expressing GLP-1 receptor.

FIG. 3 is a graph showing the in vivo activity of the heterogeneousPEG₃₀-FMS-OXM and the three PEG₃₀-FMS-OXM variants (amino, Lys12 andLys30) in the IPGTT model. All the compounds induced glucose tolerancecompared to vehicle group.

FIG. 4 shows the effect of the heterogeneous PEG30-FMS-OXM and the threePEG30-FMS-OXM variants (amino, Lys12 and Lys30) on body weight in maleob/ob mice.

FIG. 5 shows the effect of the heterogeneous PEG30-FMS-OXM and the threePEG30-FMS-OXM variants (amino, Lys12 and Lys30) on food intake in maleob/ob mice.

FIGS. 6A and 6B show the effect of the heterogeneous PEG30-FMS-OXM andthe three PEG30-FMS-OXM variants (amino, Lys12 and Lys30) on non-fasting(FIG. 6A) and fasting (FIG. 6B) glucose in male ob/ob mice.

FIG. 7 shows the effect of MOD-6031, OXM and liraglutide on cumulativefood intake in male ob/ob mice.

FIG. 8 shows the effect of MOD-6031, OXM and liraglutide on body weightin male ob/ob mice.

FIGS. 9A and 9B show the effect of MOD-6031, OXM and liraglutide onfreely feeding (FIG. 9A) and fasted plasma glucose (FIG. 9B) in maleob/ob mice.

FIGS. 10A and 10B show the effect of MOD-6031 and pair fed group onglucose tolerance (2 g/kg po) on day 2 of the study, in male ob/ob mice(FIG. 10A: plasma glucose; FIG. 10B: plasma insulin).

FIGS. 11A and 11B show the effect of MOD-6031 and pair fed group onglucose tolerance (2 g/kg po) on day 30 of the study, in male ob/ob mice(FIG. 11A: plasma glucose; FIG. 11B: plasma insulin).

FIG. 12 shows the effect of MOD-6031, OXM and liraglutide on terminalplasma cholesterol in male ob/ob mice

FIG. 13 shows the effect of PEG-FMOC-OXM, MOD 6031, and PEG-EMCS-OXM onbody weight in male ob/ob mice.

FIG. 14 shows the effect of FMOC-OXM, MOD 6031, and PEG-EMCS-OXM oncumulative food intake in male ob/ob mice.

FIGS. 15A and 15B show the effect of repeated administration ofFMOC-OXM, MOD 6031, and PEG-EMCS-OXM on plasma glucose in male ob/obmice (FIG. 15A: freely fed plasma glucose; FIG. 15B; fasted plasmaglucose).

FIGS. 16A and 16B show the effect of FMOC-OXM, MOD 6031, andPEG-EMCS-OXM on glucose tolerance (2 g/kg po) in male ob/ob mice (FIG.16A: freely fed plasma glucose; FIG. 16B; fasted plasma glucose).

FIGS. 17A and 17B show the effect of repeated administration ofFMOC-OXM, MOD 6031, and PEG-EMCS-OXM on glucose tolerance (2 g/kg po) inmale ob/ob mice (FIG. 17A: freely fed plasma glucose; FIG. 17B; fastedplasma glucose).

FIG. 18 shows the effect of repeated administration ofPEG30-S-MAL-Fmoc-OXM, MOD-6031, and PEG-EMCS-OXM on unfasted terminalplasma lipids in male ob/ob mice.

FIG. 19 shows the effect of repeated administration ofPEG30-S-MAL-Fmoc-OXM, MOD-6031, and PEG-EMCS-OXM on unfasted terminalplasma fructosamine in male ob/ob mice.

FIGS. 20A-20C show mean MOD-6031 (FIG. 20A), OXM (FIG. 20C), andPEG30-S-MAL-FMS-NHS (FIG. 20B) concentrations versus time in phosphatebuffer at different pH levels. The resin attached PEG-FMS-OXM shown inthe Figure is MOD-6031 and has the structure shown in FIG. 28A. PEG-FMSin the Figure refers to PEG30-S-MAL-FMS-NHS as presented in FIG. 28B.

FIGS. 21A-21C show mean MOD-6031 (FIG. 21A), OXM (FIG. 21C), andPEG30-S-MAL-FMS-NHS (FIG. 21B) concentrations versus time in rat plasmaat different temperatures. The resin attached PEG-FMS-OXM shown in theFigure is MOD-6031 and has the structure shown in FIG. 28A. PEG-FMS inthe Figure refers to PEG30-S-MAL-FMS-NHS as presented in FIG. 28B.

FIGS. 22A-22C show mean MOD-6031 (FIG. 22A), OXM (FIG. 22C) andPEG30-S-MAL-FMS-NHS (FIG. 22B) concentrations versus time in differentplasma types. The resin attached PEG-FMS-OXM shown in the Figure isMOD-6031 and has the structure shown in FIG. 28A. PEG-FMS in the Figurerefers to PEG30-S-MAL-FMS-NHS as presented in FIG. 28B.

FIG. 23 shows degradation assays of OXM and OXM+DPPIV at pH=6.

FIG. 24 shows degradation assays of OXM and OXM+DPPIV at pH=7.

FIG. 25 shows degradation assays of MOD-6031, MOD-6031+DPPIV (1×[DPPIVconcentration] and 10× [DPPIV concentration]) at pH=6.

FIG. 26 shows degradation assays of PEG-EMCS-OXM and PEG-EMCS-OXM+DPPIVat pH=6.

FIG. 27 shows MOD-6031 dose-dependently reduced terminal glucose andmarkedly reduced insulin.

FIG. 28 shows the structure of MOD-6031 structure wherein PEG is PEG₃₀and R₂ is SO₃H on position C₂ (FIG. 28A), and the structure ofPEG30-S-MAL-FMS-NHS (FIG. 28B)

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a long-acting oxyntomodulin and methods of producingand using same. In one aspect, the invention provides a conjugatecomprising or consisting of a dual GLP-1/Glucagon receptor agonist, apolyethylene glycol polymer (PEG polymer) and a flexible linker.

In another embodiment, this invention provides a conjugate comprising orconsisting of a dual GLP-1/Glucagon receptor agonist, a polyethyleneglycol polymer (PEG polymer) and optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) linker. In another embodiment, the invention provides a conjugatecomprising or consisting of an oxyntomodulin, a polyethylene glycolpolymer (PEG polymer) and optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) linker. In another embodiment, the PEG polymer is attached to alysine residue on position number twelve (Lys₁₂) of the oxyntomodulin'samino acid sequence via optionally substituted Fmoc or FMS linker. Inone embodiment, a long-acting oxyntomodulin is a conjugate comprising orconsisting of oxyntomodulin and polyethylene glycol polymer (PEGpolymer) attached to a lysine residue on position number twelve (Lys₁₂)of the oxyntomodulin's amino acid sequence via optionally substitutedFmoc or FMS linker.

In another embodiment, provided herein is a novel method for extendingthe serum half-life of peptides. This method is based on the use of aconjugate comprising a reversible attachment of a polyethylene glycol(PEG) chain to the peptide through a chemical linker (called FMS orFmoc) resulting in the slow release of the native peptide into thebloodstream. The released peptide can then also cross the blood brainbarrier to enter the central nervous system (CNS) or any other targetorgan. In one embodiment, the unique chemical structure of the FMSlinker leads to a specific rate of peptide release.

Hence, in another embodiment, provided herein is a method for extendingthe biological half-life of an OXM peptide. In another embodiment,provided herein is a method for extending the circulating time in abiological fluid of OXM, wherein said circulating time is extended bythe slow release of the intact OXM peptide. In another embodiment,extending said biological half-life or said circulating time of said OXMpeptide allows said OXM to cross the blood brain barrier and target theCNS. It will be well appreciated by the skilled artisan that thebiological fluid may be blood, sera, cerebrospinal fluid (CSF), and thelike.

In one embodiment, upon administration of the PEGylated oxyntomodulinconjugate of the present invention into a subject, the oxyntomodulin isreleased into a biological fluid in the subject as a result of chemicalhydrolysis of said FMS or said Fmoc linker from said conjugate. Inanother embodiment, the released oxyntomodulin is intact and regainscomplete GLP-1 and glucagon receptor binding activity. In anotherembodiment, chemically hydrolyzing said FMS or said Fmoc extends thecirculating time of said OXM peptide in said biological fluid. Inanother embodiment, extending the circulating time of said OXM allowssaid OXM to cross the blood brain barrier and target the CNS. In anotherembodiment, extending the circulating time of said OXM allows said OXMto cross the blood brain bather and target the hypothalamus. In anotherembodiment, extending the circulating time of said OXM allows said OXMto cross the blood brain barrier and target the arcuate nucleus.

In one aspect, the amino variant of PEG30-FMS-OXM is a site directedconjugate comprising OXM and mPEG(30)-SH linked through a bi-functionallinker (FMS or Fmoc). In another embodiment, the OXM peptide isconnected through its terminal amine of the N-terminus side which reactswith the N-succinimide ester (NHS) group on the linker from one sidewhile mPEG(30)-SH is connected to the maleimide moiety of the FMS linkerby its thiol group (see Examples herein). The Lys12 and Lys30 variantsare conjugated to the FMS linker through their amine group of Lysresidues. In one embodiment, the reversible-pegylation method isutilized herein to generate the long lasting oxyntomodulin (OXM)peptides provided herein (e.g. PEG30-FMS-OXM).

In one embodiment, the terms dual “GLP-1/Glucagon receptor agonist” and“agonist” are used interchangeably herein. In another embodiment, theterms also include any GLP-1/Glucagon receptor agonist known in the art.In another embodiment, the GLP-1/Glucagon receptor agonist comprises anaturally occurring dual agonist. In another embodiment, theGLP-1/Glucagon receptor agonist comprises a non-naturally occurring dualagonist. In another embodiment, a non-naturally occurring GLP-1/Glucagonreceptor agonist binds to a GLP-1 and a glucagon receptor with differentaffinities to these receptors than oxyntomodulin. In another embodiment,the preferred agonist is oxyntomodulin or OXM or a functional variantthereof.

In one embodiment, the term “functional” refers to the ability of theagonist or OXM provided herein to have biological activity, whichinclude but is not limited to, reducing weight, increasing insulinsensitivity, reducing insulin resistance, increasing energy expenditureinducing glucose tolerance, inducing glycemic control, improvingcholesterol levels, etc., as further provided herein.

In one embodiment, the invention provides a conjugate comprising anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) andoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) linker, wherein the PEG polymeris attached to a lysine residue on position number thirty (Lys₃₀) ofsaid oxyntomodulin amino acid sequence via optionally substituted Fmocor FMS linker. In one embodiment, a long-acting oxyntomodulin is aconjugate comprising or consisting of oxyntomodulin and polyethyleneglycol polymer (PEG polymer) attached to a lysine residue on positionnumber twelve (Lys₃₀) of the oxyntomodulin amino acid sequence viaoptionally substituted Fmoc or FMS linker.

In one embodiment, the invention provides a conjugate consisting of anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) andoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) linker, wherein the PEG polymeris attached to a lysine residue on position number thirty (Lys₃₀) ofsaid oxyntomodulin's amino acid sequence via optionally substituted Fmocor FMS linker. In one embodiment, a long-acting oxyntomodulin is aconjugate comprising or consisting of oxyntomodulin and polyethyleneglycol polymer (PEG polymer) attached to a lysine residue on positionnumber twelve (Lys₃₀) of the oxyntomodulin's amino acid sequence viaoptionally substituted Fmoc or FMS linker.

In one embodiment, the invention provides a conjugate comprising anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) and anoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) linker, wherein the PEG polymeris attached to the amino terminus of said oxyntomodulin via optionallysubstituted Fmoc or FMS linker. In one embodiment, a long-actingoxyntomodulin is a composition comprising or consisting of oxyntomodulinand polyethylene glycol polymer (PEG polymer) attached to the aminoterminus of the oxyntomodulin's amino acid sequence via Fmoc or FMSlinker.

In one embodiment, the invention provides a conjugate consisting of anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) linker, wherein the PEG polymer is attached to the amino terminusof said oxyntomodulin via Fmoc or FMS linker. In one embodiment, along-acting oxyntomodulin is a conjugate comprising or consisting ofoxyntomodulin and polyethylene glycol polymer (PEG polymer) attached tothe amino terminus of the oxyntomodulin's amino acid sequence via Fmocor FMS linker.

In another embodiment, the present invention provides a conjugatecomprising an oxyntomodulin peptide, and a polyethylene glycol (PEG)polymer conjugated to the oxyntomodulin peptide's lysine amino acid onposition twelve (Lys12) or position 30 (Lys30) or on the amino terminusof the oxyntomodulin peptide via a 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) linker. In another embodiment,the present invention provides a modified oxyntomodulin peptideconsisting of an oxyntomodulin peptide, and a polyethylene glycol (PEG)polymer conjugated to the oxyntomodulin peptide's lysine amino acid onposition twelve (Lys12) or position 30 (Lys30) or on the amino terminusof the oxyntomodulin peptide via a 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) linker. In another embodiment,the conjugate where PEG is attached to oxyntomodulin at Lys12, Lys30 orat the amino terminus are respectively referred to as the “Lys12variant,” the “Lys30 variant” or the “amino variant,” of oxyntomodulin.In one embodiment, the terms “amino variant” or “amino-terminus variant”are synonymous with “N-terminal variant”, “N′ variant” or “N-terminusvariant”. It is to be understood that a skilled artisan may be guided bythe present invention to readily insert lysine residues in asite-specific or random manner throughout the OXM sequence in order toattach a linker (Fmoc or FMS)/PEG conjugate provided herein at theselysine residues. In one embodiment, variants where one or more lysineresidues are located in different positions throughout the OXM sequenceand are used for conjugating OXM to PEG and cleavable linker (e.g. FMSor Fmoc), are also encompassed in the present invention.

In one embodiment, the present invention provides a conjugate comprisingan oxyntomodulin peptide, and a polyethylene glycol (PEG) polymerconjugated to the oxyntomodulin peptide's lysine amino acid on positiontwelve (Lys12) and position 30 (Lys30) via an optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) linker. In another embodiment, the present invention provides aconjugate comprising an oxyntomodulin peptide, and a polyethylene glycol(PEG) polymer conjugated to the oxyntomodulin peptide's lysine aminoacid on position twelve (Lys12) and on the amino terminus via anoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) linker. In another embodiment,the present invention provides a conjugate comprising an oxyntomodulinpeptide, and a polyethylene glycol (PEG) polymer conjugated to theoxyntomodulin peptide's lysine amino acid on position thirty (Lys30) andon the amino terminus via an optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) linker.

In another embodiment, a long-acting oxyntomodulin is a pegylatedoxyntomodulin. In another embodiment, a long-acting oxyntomodulin is areversed pegylated oxyntomodulin. In another embodiment, the phrases“long-acting oxyntomodulin,” “reversed pegylated oxyntomodulin,”“reversible PEGylated OXM,” or “a conjugate comprising or consisting ofoxyntomodulin, polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS)” are used interchangeably. In another embodiment, a long-actingoxyntomodulin is OXM linked to PEG via optionally substituted Fmoc orFMS linker. In another embodiment, the long-acting OXM is linked tooptionally substituted Fmoc or FMS via its Lys12 residue, or its Lys30residue or its amino (N′) terminus.

In one embodiment, a long-acting oxyntomodulin of the inventioncomprises a PEG polymer. In another embodiment, a long-actingoxyntomodulin of the invention comprises a PEG polymer conjugated to theamino terminus of an oxyntomodulin peptide via optionally substitutedFmoc or FMS. In another embodiment, a long-acting oxyntomodulin of theinvention comprises a PEG polymer conjugated via optionally substitutedFmoc or FMS to lysine residues 12 or 30 of the oxyntomodulin peptide. Inanother embodiment, a long-acting oxyntomodulin of the inventioncomprises a PEG polymer conjugated via optionally substituted Fmoc orFMS to both the amino terminus of an oxyntomodulin peptide and to lysineresidues 12 and 30 of oxyntomodulin.

In another embodiment, a long-acting oxyntomodulin is a conjugatecomprising or consisting of oxyntomodulin, polyethylene glycol polymer(PEG polymer) and optionally substituted 9-fluorenylmethoxycarbonyl(Fmoc) or sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of1:0.2-10:0.2-10. In another embodiment, a long-acting oxyntomodulin is aconjugate comprising or consisting of oxyntomodulin, polyethylene glycolpolymer (PEG polymer) and 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of1:0.5-2:0.5-2. In another embodiment, a long-acting oxyntomodulin is aconjugate comprising or consisting of oxyntomodulin, polyethylene glycolpolymer (PEG polymer) and optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS) in a molar ratio of 1:1:1. In another embodiment, a long-actingoxyntomodulin includes a PEG polymer conjugated to the amino terminus ofoxyntomodulin via optionally substituted Fmoc or FMS. In anotherembodiment, the molar ratio of OXM-PEG- and linker is 1:1:1-1:1:3.5. Inanother embodiment, the molar ratio is 1:1:1-1:1:10.0. In anotherembodiment, the higher ratio of linker allows for optimized yield of theconjugate.

In another embodiment, a long-acting oxyntomodulin is linked to PEG viaa reversible linker such as, but not limited to, optionally substitutedFmoc and FMS. In another embodiment, Fmoc and FMS are sensitive to basesand are removable under physiological conditions. In another embodiment,a reversible linker is a linker that is sensitive to bases and isremovable under physiological conditions. In another embodiment, areversible linker is a linker that is sensitive to bases and isremovable under physiological conditions in the blood, plasma, or lymph.In another embodiment, a reversible linker is a linker that is sensitiveto bases and is removable under physiological conditions in a bodyfluid. In another embodiment, a reversible linker is a linker that isremovable in a body fluid having a basic pH. In another embodiment, alinker that is sensitive to bases is cleaved upon exposure to a basicenvironment thus releasing OXM from the linker and PEG. In anotherembodiment, a linker that is sensitive to temperature is cleaved uponexposure to specific temperature that allows for such cleavage to takeplace. In another embodiment, the temperature that enables cleavage ofthe linker is within the physiological range. In another embodiment, areversible linker is any reversible linker known in the art.

In another embodiment, a reverse pegylated oxyntomodulin is a conjugatewherein OXM is linked to PEG via a reversible linker. In anotherembodiment, a reverse pegylated oxyntomodulin releases free OXM uponexposure to a basic environment. In another embodiment, a reversepegylated oxyntomodulin releases free OXM upon exposure to blood orplasma. In another embodiment, a long-acting oxyntomodulin comprises PEGand oxyntomodulin that are not linked directly to each other, as instandard pegylation procedures, but rather both residues are linked todifferent positions of Fmoc or FMS which are highly sensitive to basesand are removable under regular physiological conditions. In anotherembodiment, regular physiological conditions include a physiologicenvironment such as the blood or plasma.

In another embodiment, the structures and the processes of making Fmocand FMS are described in U.S. Pat. No. 7,585,837. The disclosure of U.S.Pat. No. 7,585,837 is hereby incorporated by reference in its entirety.

In one embodiment, the conjugate of this invention is presented by thestructure of formula I:(X)n-Y,wherein Y is a dual GLP-1/Glucagon receptor agonist bearing a freeamino, carboxyl, or hydroxyl;

X is a radical of formula (i):

wherein R₁ is a radical containing a protein or polymer carrier moiety;polyethylene glycol (PEG) moiety;

R₂ is selected from the group consisting of hydrogen, alkyl, alkoxy,alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, —SO₃H, —SO₂NHR,amino, ammonium, carboxyl, PO₃H2, and OPO₃H₂;

R is selected from the group consisting of hydrogen, alkyl and aryl;

R₃ and R₄, the same or different, are each selected from the groupconsisting of hydrogen, alkyl and aryl;

A is a covalent bond when the radical is linked to an amino or hydroxylgroup of the OXM-Y; and

n is an integer of at least one, and pharmaceutically acceptable saltsthereof.

In one embodiment, R₁ is a radical containing a protein or polymercarrier moiety; polyethylene glycol (PEG) moiety. In another embodiment,the PEG moiety is —NH—C(O)—(CH₂)p-maleimide-S-PEG, wherein p is aninteger between 1-6. In another embodiment, p is 2.

In another embodiment, n of formula I is an integer of at least 1. Inanother embodiment, n is 1. In another embodiment, n is 2. In anotherembodiment, n is between 1 to 5. In another embodiment, n is between 2to 5.

In another embodiment, the GLP-1/Glucagon receptor agonist isoxyntomodulin (OXM).

In another embodiment, the terms “alkyl”, “alkoxy”, “alkoxyalkyl”,“aryl”, “alkaryl” and “aralkyl” are used to denote alkyl radicals of1-8, preferably 1-4 carbon atoms, e.g. methyl, ethyl, propyl, isopropyland butyl, and aryl radicals of 6-10 carbon atoms, e.g. phenyl andnaphthyl. The term “halogen” includes bromo, fluoro, chloro and iodo.

In another embodiment, R₂, R₃ and R₄ are each hydrogen.

In another embodiment R₂ is -hydrogen, A is —OCO—[—OC(═O)—], R₃ and R₄are each hydrogen, namely the 9-fluorenylmethoxycarbonyl radical(hereinafter “Fmoc”).

In another embodiment, R₂ is —SO₃H at position 2 of the fluorene ring,R₃ and R₄ are each hydrogen, and A is —OCO—[—OC(═O)—]. In anotherembodiment, R₂ is —SO₃H at position 1 of the fluorene ring, R₃ and R₄are each hydrogen, and A is —OCO—[—OC(═O)]. In another embodiment, R₂ is—SO₃H at position 3 of the fluorene ring, R₃ and R₄ are each hydrogen,and A is —OCO—[—OC(═O)]. In another embodiment, R₂ is —SO₃H at position4 of the fluorene ring, R₃ and R₄ are each hydrogen, and A is—OCO—[—OC(═O)]. In another embodiment, SO₃H is at position, 1, 2, 3 or 4of the fluorene or any combination thereof.

In one embodiment, the conjugate of this invention is presented by thestructure of formula II, wherein OXM is linked to the linker via theamino-terminal of said OXM:

wherein R₂ is hydrogen or SO₃H. In one embodiment, R₂ is SO₃H and is atposition 2 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 1 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 3 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 4 of the fluorene. In another embodiment, SO₃H is at position,1, 2, 3 or 4 of the fluorene or combination thereof. In one embodiment,R₂ is SO₃H and is at position 2 of the fluorene and the PEG is PEG30. Inanother embodiment, R₂ is SO₃H and is at position 1 of the fluorine andthe PEG is PEG30. In another embodiment, R₂ is SO₃H and is at position 3of the fluorine and the PEG is PEG30. In another embodiment, R₂ is SO₃Hand is at position 4 of the fluorine and the PEG is PEG30.

In one embodiment, MOD-6031 is presented by the structure of formulaIIa, wherein PEG is PEG₃₀ and R₂ is SO₃H at position 2 of the fluorene:

In one embodiment, the conjugate of this invention is presented by thestructure of formula III, wherein OXM is linked to the linker via theamino residue of Lys₃₀ of said OXM:

wherein R₂ is hydrogen or SO₃H. In one embodiment, R₂ is SO₃H and is atposition 2 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 1 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 3 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 4 of the fluorene. In another embodiment, SO₃H is at position,1, 2, 3 or 4 of the fluorene or any combination thereof.

In one embodiment, the conjugate of this invention is presented by thestructure of formula IV wherein OXM is linked to the linker via theamino residue of Lys12 of said OXM:

wherein R₂ is hydrogen or SO₃H. In one embodiment, R₂ is SO₃H and is atposition 2 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 1 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 3 of the fluorene. In another embodiment, R₂ is SO₃H and is atposition 4 of the fluorene. In another embodiment, SO₃H is at position,1, 2, 3 or 4 of the fluorene or any combination thereof.

In one embodiment, the conjugate of this invention is presented by theformula: PEG-S-MAL-Fmoc-OXM, PEG-S-MAL-FMS-OXM, (PEG-S-MAL-FMS)n-OXM or(PEG-S-MAL-Fmoc)n-OXM; wherein n is an integer of at least 1. In anotherembodiment, the OXM in linked to the FMS or Fmoc via amino terminal ofthe OXM or amino residue of one of OXM amino acids. In anotherembodiment, the PEG is linked to the Fmoc or FMSvia—NH—C(O)—(CH₂)p-maleimide-S— wherein p is an integer between 1-6, andwherein the PEG is linked to the sulfide group.

In one embodiment, Fmoc of this invention is presented by the followingstructure:

In one embodiment, FMS of this invention is presented by the followingstructure:

In one embodiment, R₂ is SO₃H and is at position 2 of the fluorene. Inanother embodiment, R₂ is SO₃H and is at position 1 of the fluorene. Inanother embodiment, R₂ is SO₃H and is at position 3 of the fluorene. Inanother embodiment, R₂ is SO₃H and is at position 4 of the fluorene. Inanother embodiment, SO₃H is at position, 1, 2, 3 or 4 of the fluorene orany combination thereof.

In another embodiment, OXM comprises the amino acid sequence of SEQ IDNO: 1. In another embodiment, OXM consists of the amino acid sequence ofSEQ ID NO: 1. In another embodiment, SEQ ID NO: 1 comprises or consistsof the following amino acid (AA) sequence:HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO: 1). In anotherembodiment, OXM comprises or consists of the amino acid sequencedepicted in CAS No. 62340-29-8.

In another embodiment, OXM is human OXM or any mammal OXM. In anotherembodiment, OXM is also referred to as glucagon-37 or bioactiveenteroglucagon. In another embodiment, OXM is a dual GLP-1/Glucagonreceptor agonist. In another embodiment, OXM is a biologically activefragment of OXM. In another embodiment, biologically active OXM extendsfrom amino acid 30 to amino acid 37 of SEQ ID NO: 1. In anotherembodiment, biologically active OXM extends from amino acid 19 to aminoacid 37 of SEQ ID NO: 1. In another embodiment, OXM of the inventioncorresponds to an octapeptide from which the two C-terminal amino acidsare deleted. In another embodiment, OXM of the invention corresponds toany fragment of SEQ ID NO: 1 which retains OXM activity as providedherein.

In one embodiment, OXM refers to a peptide homologue of the peptide ofSEQ ID NO: 1. In one embodiment, OXM amino acid sequence of the presentinvention is at least 50% homologous to the OXM sequence set forth inSEQ ID NO: 1 as determined using BlastP software of the National Centerof Biotechnology Information (NCBI) using default parameters. In oneembodiment, OXM amino acid sequence of the present invention is at least60% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 70% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 80% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 90% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 95% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.

In one embodiment, the OXM of the present invention are utilized intherapeutics which requires OXM to be in a soluble form. In anotherembodiment, OXM of the present invention includes one or morenon-natural or natural polar amino acid, including, but not limited to,serine and threonine which are capable of increasing protein solubilitydue to their hydroxyl-containing side chain.

In one embodiment, OXM of present invention is biochemically synthesizedsuch as by using standard solid phase techniques. In another embodiment,these biochemical methods include exclusive solid phase synthesis,partial solid phase synthesis, fragment condensation, or classicalsolution synthesis.

In one embodiment, solid phase OXM synthesis procedures are well knownto one skilled in the art and further described by John Morrow Stewartand Janis Dillaha Young, Solid Phase Protein Syntheses (2nd Ed., PierceChemical Company, 1984). In another embodiment, synthetic proteins arepurified by preparative high performance liquid chromatography[Creighton T. (1983) Proteins, structures and molecular principles. WHFreeman and Co. N.Y.] and the composition of which can be confirmed viaamino acid sequencing by methods known to one skilled in the art.

In another embodiment, recombinant protein techniques are used togenerate the OXM of the present invention. In some embodiments,recombinant protein techniques are used for the generation of largeamounts of the OXM of the present invention. In another embodiment,recombinant techniques are described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 andBrogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.

In another embodiment, OXM of the present invention is synthesized usinga polynucleotide encoding OXM of the present invention. In someembodiments, the polynucleotide encoding OXM of the present invention isligated into an expression vector, comprising a transcriptional controlof a cis-regulatory sequence (e.g., promoter sequence). In someembodiments, the cis-regulatory sequence is suitable for directingconstitutive expression of the OXM of the present invention.

In one embodiment, the phrase “a polynucleotide” refers to a single ordouble stranded nucleic acid sequence which be isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

In one embodiment, “complementary polynucleotide sequence” refers to asequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.In one embodiment, the sequence can be subsequently amplified in vivo orin vitro using a DNA polymerase.

In one embodiment, “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

In one embodiment, “composite polynucleotide sequence” refers to asequence, which is at least partially complementary and at leastpartially genomic. In one embodiment, a composite sequence can includesome exonal sequences required to encode the peptide of the presentinvention, as well as some intronic sequences interposing there between.In one embodiment, the intronic sequences can be of any source,including of other genes, and typically will include conserved splicingsignal sequences. In one embodiment, intronic sequences include cisacting expression regulatory elements.

In one embodiment, polynucleotides of the present invention are preparedusing PCR techniques, or any other method or procedure known to oneskilled in the art. In some embodiments, the procedure involves theligation of two different DNA sequences (See, for example, “CurrentProtocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons,1992). In one embodiment, a variety of prokaryotic or eukaryotic cellscan be used as host-expression systems to express the OXM of the presentinvention. In another embodiment, these include, but are not limited to,microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the protein coding sequence; yeast transformed withrecombinant yeast expression vectors containing the protein codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors, such asTi plasmid, containing the protein coding sequence.

In one embodiment, non-bacterial expression systems are used (e.g.mammalian expression systems such as CHO cells) to express the OXM ofthe present invention. In one embodiment, the expression vector used toexpress polynucleotides of the present invention in mammalian cells ispCI-DHFR vector comprising a CMV promoter and a neomycin resistancegene.

In another embodiment, in bacterial systems of the present invention, anumber of expression vectors can be advantageously selected dependingupon the use intended for the protein expressed. In one embodiment,large quantities of OXM are desired. In one embodiment, vectors thatdirect the expression of high levels of the protein product, possibly asa fusion with a hydrophobic signal sequence, which directs the expressedproduct into the periplasm of the bacteria or the culture medium wherethe protein product is readily purified are desired. In one embodiment,certain fusion protein engineered with a specific cleavage site to aidin recovery of the protein. In one embodiment, vectors adaptable to suchmanipulation include, but are not limited to, the pET series of E. coliexpression vectors [Studier et al., Methods in Enzymol. 185:60-89(1990)].

In one embodiment, yeast expression systems are used. In one embodiment,a number of vectors containing constitutive or inducible promoters canbe used in yeast as disclosed in U.S. Pat. No. 5,932,447. In anotherembodiment, vectors which promote integration of foreign DNA sequencesinto the yeast chromosome are used.

In one embodiment, the expression vector of the present invention canfurther include additional polynucleotide sequences that allow, forexample, the translation of several proteins from a single mRNA such asan internal ribosome entry site (IRES) and sequences for genomicintegration of the promoter-chimeric protein.

In one embodiment, mammalian expression vectors include, but are notlimited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB,pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

In another embodiment, expression vectors containing regulatory elementsfrom eukaryotic viruses such as retroviruses are used by the presentinvention. SV40 vectors include pSVT7 and pMT2. In another embodiment,vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Bar virus include pHEBO, and p2O5. Otherexemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

In one embodiment, plant expression vectors are used. In one embodiment,the expression of OXM coding sequence is driven by a number ofpromoters. In another embodiment, viral promoters such as the 35S RNAand 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514(1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J.6:307-311 (1987)] are used. In another embodiment, plant promoters areused such as, for example, the small subunit of RUBISCO [Coruzzi et al.,EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843(1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B[Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment,constructs are introduced into plant cells using Ti plasmid, Ri plasmid,plant viral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach [Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Otherexpression systems such as insects and mammalian host cell systems,which are well known in the art, can also be used by the presentinvention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the protein), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressed protein.

Various methods, in some embodiments, can be used to introduce theexpression vector of the present invention into the host cell system. Insome embodiments, such methods are generally described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (1989, 1992), in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Changet al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vegaet al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

In one embodiment, transformed cells are cultured under effectiveconditions, which allow for the expression of high amounts ofrecombinant OXM. In another embodiment, effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Inone embodiment, an effective medium refers to any medium in which a cellis cultured to produce the recombinant OXM of the present invention. Inanother embodiment, a medium typically includes an aqueous solutionhaving assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. In one embodiment, cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes and petri plates. In another embodiment,culturing is carried out at a temperature, pH and oxygen contentappropriate for a recombinant cell. In another embodiment, culturingconditions are within the expertise of one of ordinary skill in the art.

In one embodiment, depending on the vector and host system used forproduction, resultant OXM of the present invention either remain withinthe recombinant cell, secreted into the fermentation medium, secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or retained on the outer surface of a cell or viralmembrane.

In one embodiment, following a predetermined time in culture, recoveryof the recombinant OXM is affected.

In one embodiment, the phrase “recovering the recombinant OXM” usedherein refers to collecting the whole fermentation medium containing theOXM and need not imply additional steps of separation or purification.

In another embodiment, the OXM provided herein can be chemicallymodified. In particular, the amino acid side chains, the amino terminusand/or the carboxy acid terminus of OXM can be modified. For example,the OXM can undergo one or more of alkylation, disulphide formation,metal complexation, acylation, esterification, amidation, nitration,treatment with acid, treatment with base, oxidation or reduction.Methods for carrying out these processes are well known in the art. Inparticular the OXM is provided as a lower alkyl ester, a lower alkylamide, a lower dialkyl amide, an acid addition salt, a carboxylate saltor an alkali addition salt thereof. In particular, the amino orcarboxylic termini of the OXM may be derivatised by for example,esterification, amidation, acylation, oxidation or reduction. Inparticular, the carboxylic terminus of the OXM can be derivatised toform an amide moiety.

In another embodiment, modifications include, but are not limited to Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH₂—NH, CH₂—S, CH₂—S═O,O═C—NH, CH₂—O, CH₂—CH₂, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

In another embodiment, peptide bonds (—CO—NH—) within the peptide aresubstituted. In some embodiments, the peptide bonds are substituted byN-methylated bonds (—N(CH3)-CO—). In another embodiments, the peptidebonds are substituted by ester bonds (—C(R)H—C—O—O—C(R)—N—). In anotherembodiment, the peptide bonds are substituted by ketomethylen bonds(—CO—CH2-). In another embodiment, the peptide bonds are substituted byα-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carbabonds (—CH2-NH—). In another embodiments, the peptide bonds aresubstituted by hydroxyethylene bonds (—CH(OH)—CH2-). In anotherembodiment, the peptide bonds are substituted by thioamide bonds(—CS—NH—). In some embodiments, the peptide bonds are substituted byolefinic double bonds (—CH═CH—). In another embodiment, the peptidebonds are substituted by retro amide bonds (—NH—CO—). In anotherembodiment, the peptide bonds are substituted by peptide derivatives(—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturallypresented on the carbon atom. In some embodiments, these modificationsoccur at any of the bonds along the peptide chain and even at several(2-3 bonds) at the same time.

In one embodiment, natural aromatic amino acids of the protein such asTrp, Tyr and Phe, are substituted for synthetic non-natural acid such asPhenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivativesof Phe, halogenated derivatives of Phe or o-methyl-Tyr. In anotherembodiment, the peptides of the present invention include one or moremodified amino acid or one or more non-amino acid monomers (e.g. fattyacid, complex carbohydrates etc).

In comparison to the wild-type OXM, the OXM derivatives or variants ofthe present invention contain several amino acid substitutions, and/orcan be PEGylated or otherwise modified (e.g. recombinantly orchemically).

The OXM provided herein also covers any analogue of the above OXMsequence. Any one or more amino acid residues in the sequence can beindependently replaced with a conservative replacement as well known inthe art i.e. replacing an amino acid with one of a similar chemical typesuch as replacing one hydrophobic amino acid with another.Alternatively, non-conservative amino acid mutations can be made thatresult in an enhanced effect or biological activity of OXM. In oneembodiment, the OXM is modified to be resistant to cleavage andinactivation by dipeptidyl peptidase IV (DPP-IV). Derivatives, andvariants of OXM and methods of generating the same are disclosed in USPatent Application Publication Nos. 2011/0152182, US Patent ApplicationPublication Nos. 2011/0034374, US Patent Application Publication Nos.2010/0144617, all of which are incorporated by reference herein.

In one embodiment, “amino acid” or “amino acids” is understood toinclude the 20 naturally occurring amino acids; those amino acids oftenmodified post-translationally in vivo, including, for example,hydroxyproline, phosphoserine and phosphothreonine; and other unusualamino acid including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodemosine, nor-valine, nor-leucine and ornithine. Inone embodiment, “amino acid” includes both D- and L-amino acids. It isto be understood that other synthetic or modified amino acids can bealso be used.

In one embodiment, oxyntomodulin (OXM) of the present invention ispurified using a variety of standard protein purification techniques,such as, but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, concanavalin A chromatography, chromatofocusing anddifferential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the protein of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the protein can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the protein and the cleavable moiety and the protein can bereleased from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)]. In anotherembodiment, the OXM of the present invention is retrieved in“substantially pure” form. In another embodiment, the phrase“substantially pure” refers to a purity that allows for the effectiveuse of the OXM in the applications described herein.

In one embodiment, the OXM of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In another embodiment, in vitro binding activity is ascertained bymeasuring the ability of native, recombinant and/or reverse pegylatedOXM as described herein as well as pharmaceutical compositionscomprising the same to treat or ameliorate diseases or conditions suchas but not limited to: diabetes mellitus, obesity, eating disorders,metabolic disorders, etc. In another embodiment, in vivo activity isdeduced by known measures of the disease that is being treated.

In another embodiment, the molar ratio of OXM-PEG- and linker is1:1:1-1:1:3.5. In another embodiment, the molar ratio is 1:1:1-1:1:10.0.In another embodiment, the higher ratio of linker allows for optimizedyield of the composition.

In another embodiment, a PEG polymer is attached to the amino terminusor lysine residue of oxyntomodulin via optionally substituted Fmoc orFMS. In another embodiment, the terms “attached” and “linked” are useinterchangeably. In another embodiment, the PEG polymer is linked to theα-amino side chain of OXM. In another embodiment, the PEG polymer islinked to the ε-amino side chain of OXM. In another embodiment, the PEGpolymer is linked to one or more ε-amino side chain of OXM. In anotherembodiment, the PEG polymer comprises a sulfhydryl moiety.

In another embodiment, PEG is linear. In another embodiment, PEG isbranched. In another embodiment, PEG has a molecular weight in the rangeof 200 to 200,000 Da. In another embodiment, PEG has a molecular weightin the range of 5,000 to 80,000 Da. In another embodiment, PEG has amolecular weight in the range of 5,000 to 40,000 Da. In anotherembodiment, PEG has a molecular weight in the range of 20,000 Da to40,000 Da. In one embodiment, PEG₃₀ refers to a PEG with an averagemolecular weight of 30,000 Da. PEG₄₀ refers to a PEG with an averagemolecular weight of 40,000 Da.

Biological Activity

In another embodiment, reverse pegylation OXM of this invention rendersOXM a long-acting OXM. In another embodiment, long-acting oxyntomodulinis an oxyntomodulin with an extended biological half-life. In anotherembodiment, reverse pegylation provides protection against degradationof OXM. In another embodiment, reverse pegylation provides protectionagainst degradation of OXM by DPPIV. In another embodiment, reversepegylation effects the C_(max) of OXM and reduces side effectsassociated with administration of the conjugate provided herein. Inanother embodiment, reverse pegylation extends the T_(max) of OXM. Inanother embodiment, reverse pegylation extends the circulatory half-liveof OXM. In another embodiment, reverse pegylated OXM has improvedbioavailability compared to non-modified OXM. In another embodiment,reverse pegylated OXM has improved biological activity compared tonon-modified OXM. In another embodiment, reverse pegylation enhances thepotency of OXM. In another embodiment, reverse pegylated OXM hasimproved insulin sensitivity. In another embodiment, reverse pegylatedOXM dose-dependently decreases terminal glucose. In another embodiment,reverse pegylated OXM dose-dependently decreases insulin.

In other embodiments, a reverse pegylated OXM of this invention is atleast equivalent to the non-modified OXM, in terms of biochemicalmeasures. In other embodiments, a reverse pegylated OXM is at leastequivalent to the non-modified OXM, in terms of pharmacologicalmeasures. In other embodiments, a reverse pegylated OXM is at leastequivalent to the non-modified OXM, in terms of binding capacity (Kd).In other embodiments, a reverse pegylated OXM is at least equivalent tothe non-modified OXM, in terms of absorption through the digestivesystem. In other embodiments, a reverse pegylated OXM is more stableduring absorption through the digestive system than non-modified OXM.

In another embodiment, a reverse pegylated OXM of this inventionexhibits improved blood area under the curve (AUC) levels compared tofree OXM. In another embodiment, a reverse pegylated OXM exhibitsimproved biological activity and blood area under the curve (AUC) levelscompared to free OXM. In another embodiment, a reverse pegylated OXMexhibits improved blood retention time (t_(1/2)) compared to free OXM.In another embodiment, a reverse pegylated OXM exhibits improvedbiological activity and blood retention time (t_(1/2)) compared to freeOXM. In another embodiment, a reverse pegylated OXM exhibits improvedblood C_(max) levels compared to free OXM, where in another embodimentit results in a slower release process that reduces side effectsassociated with administration of the reverse pegylated compositionsprovided herein. In another embodiment, a reverse pegylated OXM exhibitsimproved biological activity and blood C_(max) levels compared to freeOXM. In another embodiment, provided herein a method of improving OXM'sAUC, C_(max), t_(1/2), biological activity, or any combination thereofcomprising or consisting of the step of conjugating a polyethyleneglycol polymer (PEG polymer) to the amino terminus of free OXM via9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, improvement of OXM's AUC, C_(max), t_(1/2),biological activity, or any combination thereof by conjugating apolyethylene glycol polymer (PEG polymer) to the amino terminus of freeOXM via optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) enables the reduction in dosingfrequency of OXM. In another embodiment, provided herein a method forreducing a dosing frequency of OXM, comprising or consisting of the stepof conjugating a polyethylene glycol polymer (PEG polymer) to the aminoterminus or lysine residues of OXM via optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS). In another embodiment, reverse pegylation of OXM of thisinvention is advantageous in permitting lower dosages to be used. In oneembodiment, the long-acting OXM of the invention maintains thebiological activity of unmodified OXM. In another embodiment, thelong-acting OXM of the invention comprising OXM biological activity. Inanother embodiment, the biological activity of a long-acting OXM of theinvention comprises reducing digestive secretions. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises reducing and delaying gastric emptying. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises the inhibition of the fed motility pattern in thesmall intestine. In another embodiment, the biological activity of along-acting OXM of the invention comprises the inhibition of acidsecretion stimulated by pentagastrin. In another embodiment, thebiological activity of a long-acting OXM of the invention comprises anincrease of gastric somatostatin release. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisespotentiating the effects of peptide YY. In another embodiment, thebiological activity of a long-acting OXM of the invention comprises theinhibition of ghrelin release. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises the stimulationof aminopyrine accumulation and cAMP production. In another embodiment,the biological activity of a long-acting OXM of the invention comprisesbinding the GLP-1 receptor. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises binding theGlucagon receptor. In another embodiment, the biological activity of along-acting OXM of the invention comprises stimulating H+ production byactivating the adenylate cyclase. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises inhibitinghistamine-stimulated gastric acid secretion. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisesinhibiting food intake. In another embodiment, the biological activityof a long-acting OXM of the invention comprises stimulating insulinrelease. In another embodiment, the biological activity of a long-actingOXM of the invention comprises inhibiting exocrine pancreatic secretion.In another embodiment, the biological activity of a long-acting OXM ofthe invention comprises increasing insulin sensitivity. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises reducing glucose levels. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisesreducing terminal glucose. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises reducinginsulin.

In one embodiment, the present invention further provides a method forextending the biological half-life of oxyntomodulin, consisting of thestep of conjugating oxyntomodulin, a polyethylene glycol polymer (PEGpolymer) and optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein, in another embodiment, the PEG polymer is conjugated to aLysine residue on position number 12 or to a Lysine residue on positionnumber 30 or to the amino terminus of the oxyntomodulin's amino acidsequence via optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to a Lysine residue on positionnumber 12 of the oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to a Lysine residue on positionnumber 30 of said oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to the amino terminus of saidoxyntomodulin's amino acid sequence via optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 12 or to the Lysine residue on positionnumber 30 or to the amino terminus of the oxyntomodulin's amino acidsequence via optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the invention relates to a method of improvingthe area under the curve (AUC) of oxyntomodulin, consisting of the stepof conjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 12 of the oxyntomodulin's amino acid sequencevia optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 30 of the oxyntomodulin's amino acid sequencevia optionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the aminoterminus of the oxyntomodulin's amino acid sequence via optionallysubstituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In one aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 12 or to the Lysine residue on position number 30 or tothe amino terminus of the oxyntomodulis amino acid sequence viaoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In another aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 12 of the oxyntomodulis amino acid sequence viaoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In another aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 30 of the oxyntomodulis amino acid sequence viaoptionally substituted 9-fluorenylmethoxycarbonyl (Fmoc) orsulfo-9-fluorenylmethoxycarbonyl (FMS).

In another aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the amino terminus of theoxyntomodulis amino acid sequence via optionally substituted9-fluorenylmethoxycarbonyl (Fmoc) or sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the present invention further provides a methodfor reducing food intake, in a subject, comprising the step ofadministering a conjugate of this invention. In another embodiment, theconjugate is represented by the structure of formula I-IV.

In another embodiment, the present invention further provides a methodfor reducing body weight in a subject, comprising the step ofadministering to the subject a conjugate of this invention. In anotherembodiment, the conjugate is represented by the structure of formulaI-IV.

In another embodiment, the present invention further provides a methodfor inducing glycemic control in a subject, comprising the step ofadministering a conjugate of this invention. In another embodiment, theconjugate is represented by the structure of formula I-IV.

In another embodiment, the present invention further provides a methodfor improving glycemic and lipid profiles in a subject, comprising thestep of administering to the subject a conjugate of this invention. Inanother embodiment, the conjugate is represented by the structure offormula I-IV.

In yet another embodiment, the present invention further provides amethod for improving glycemic profile in a subject, comprising the stepof administering to the subject a conjugate of this invention. Inanother embodiment, the conjugate is represented by the structure offormula I-IV.

In an additional embodiment, the present invention further provides amethod for improving lipid profile in a subject, comprising the step ofadministering to the subjects conjugate of this invention. In anotherembodiment, the conjugate is represented by the structure of formulaI-IV.

The amino variant, for example the variant where FMS is linked to OXMvia the terminal amino group, provided herein unexpectedly achievesreduced food intake, weight control and glycemic control, as exemplifiedherein (see Example 5). In one embodiment, the PEG modification of theOXM peptide provided herein unexpectedly does not interfere with OXMfunction.

In another embodiment, the present invention provides a method forimproving cholesterol levels in a subject, comprising the step ofadministering to the subject an effective amount of a conjugate of thisinvention. In another embodiment, the conjugate is represented by thestructure of formula I-IV. In another embodiment, improving cholesterollevels comprises reducing LDL cholesterol while increasing HDLcholesterol in a subject. In another embodiment, LDL cholesterol levelsare reduced to below 200 mg/dL, but above 0 mg/dL. In anotherembodiment, LDL cholesterol levels are reduced to about 100-129 mg/dL.In another embodiment, LDL cholesterol levels are reduced to below 100mg/dL, but above 0 mg/dL. In another embodiment, LDL cholesterol levelsare reduced to below 70 mg/dL, but above 0 mg/dL. In another embodiment,LDL cholesterol levels are reduced to below 5.2 mmol/L, but above 0mmol/L. In another embodiment, LDL cholesterol levels are reduced toabout 2.6 to 3.3 mmol/L. In another embodiment, LDL cholesterol levelsare reduced to below 2.6 mmol/L, but above 0 mmol/L. In anotherembodiment, LDL cholesterol levels are reduced to below 1.8 mmol/L, butabove 0 mmol/L.

In another embodiment, the present invention further provides a methodfor reducing insulin resistance in a subject, comprising the step ofadministering to the subject an effective amount of a conjugate of thisinvention. In another embodiment, the conjugate is represented by thestructure of formula I-IV.

In another embodiment, the biological activity of a long-acting OXM ofthe invention comprises inhibiting pancreatic secretion through a vagalneural indirect mechanism. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises reducinghydromineral transport through the small intestine. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises stimulating glucose uptake. In another embodiment,the biological activity of a long-acting OXM of the invention comprisescontrolling/stimulating somatostatin secretion. In another embodiment,the biological activity of a long-acting OXM of the invention comprisesreduction in both food intake and body weight gain. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises reduction in adiposity. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisesappetite suppression. In another embodiment, the biological activity ofa long-acting OXM of the invention comprises improving glycemic andlipid profiles. In another embodiment, the biological activity of along-acting OXM of the invention comprises induction of anorexia. Inanother embodiment, the biological activity of a long-acting OXM of theinvention comprises reducing body weight in overweight and obesesubjects. In another embodiment, the biological activity of along-acting OXM of the invention comprises inducing changes in thelevels of the adipose hormones leptin and adiponectin. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises increasing energy expenditure in addition todecreasing energy intake in overweight and obese subjects. In anotherembodiment, the long-acting OXM of this invention is a conjugate offormula I-IV.

Process of Preparation

In another embodiment, a long-acting OXM of this invention is preparedusing PEGylating agents, meaning any PEG derivative which is capable ofreacting with a functional group such as, but not limited to, NH₂, OH,SH, COOH, CHO, —N═C═O, —N═C═S, —SO₂Cl, —SO₂CH═CH₂, —PO₂Cl, —(CH₂)xHal,present at the fluorene ring of the Fmoc or FMS moiety. In anotherembodiment, the PEGylating agent is usually used in itsmono-methoxylated form where only one hydroxyl group at one terminus ofthe PEG molecule is available for conjugation. In another embodiment, abifunctional form of PEG where both termini are available forconjugation may be used if, for example, it is desired to obtain aconjugate with two peptide or protein residues covalently attached to asingle PEG moiety.

In another embodiment, branched PEGs are represented as R(PEG-OH)_(m) inwhich R represents a central core moiety such as pentaerythritol orglycerol, and m represents the number of branching arms. The number ofbranching arms (m) can range from three to a hundred or more. In anotherembodiment, the hydroxyl groups are subject to chemical modification. Inanother embodiment, branched PEG molecules are described in U.S. Pat.No. 6,113,906, No. 5,919,455, No. 5,643,575, and No. 5,681,567, whichare hereby incorporated by reference in their entirety.

In another embodiment, the present invention provides OXM with a PEGmoiety which is not attached directly to the OXM, as in the standardpegylation procedure, but rather the PEG moiety is attached through alinker such as optionally substituted Fmoc or FMS. In anotherembodiment, the linker is highly sensitive to bases and is removableunder mild basic conditions. In another embodiment, OXM connected to PEGvia optionally substituted Fmoc or FMS is equivalently active to thefree OXM. In another embodiment, OXM connected to PEG via optionallysubstituted Fmoc or FMS is more active than the free OXM. In anotherembodiment, OXM connected to PEG via optionally substituted Fmoc or FMScomprises different activity than the free OXM. In another embodiment,OXM connected to PEG via optionally substituted Fmoc or FMS unlike thefree OXM, has no central nervous system activity. In another embodiment,OXM connected to PEG via optionally substituted Fmoc or FMS unlike thefree OXM, can not enter the brain through the blood brain bather. Inanother embodiment, OXM connected to PEG via Fmoc or FMS comprisesextended circulation half-life compared to the free OXM. In anotherembodiment, OXM connected to PEG via Fmoc or FMS loses its PEG moietytogether with the Fmoc or FMS moiety thus recovering the free OXM.

In another embodiment, pegylation of OXM and preparation of the(PEG-S-MAL-Fmoc)n-OXM or (PEG-S-MAL-FMS)n-OXM conjugates includesattaching MAL-FMS-NHS or MAL-Fmoc-NHS to the amine component of OXM,thus obtaining a MAL-FMS-OXM or MAL-Fmoc-OXM conjugate, and thenreacting PEG-SH with the maleimide moiety on MAL-FMS-OXM, producingPEG-S-MAL-FMS-OXM or PEG-S_MAL-Fmoc-OXM, the (PEG-S-MAL-FMS)n-OXM or(PEG-S-MAL-Fmoc)n-OXM conjugate, respectively.

In another embodiment, MAL-Fmoc-NHS is represented by the followingstructure:

In another embodiment, MAL-FMS-NHS is represented by the followingstructure

In one embodiment, SO₃H is at position 2 of the fluorene. In anotherembodiment, SO₃H is at position 1 of the fluorene. In anotherembodiment, SO₃H is at position 3 of the fluorene. In anotherembodiment, SO₃H is at position 4 of the fluorene. In anotherembodiment, SO₃H is at position, 1, 2, 3 or 4 of the fluorene or anycombination thereof.

In another embodiment, MAL-Fmoc-OXM is represented by the followingstructure:

In another embodiment, MAL-FMS-OXM is represented by the followingstructure:

In one embodiment, SO₃H is at position 2 of the fluorene. In anotherembodiment, SO₃H is at position 1 of the fluorene. In anotherembodiment, SO₃H is at position 3 of the fluorene. In anotherembodiment, SO₃H is at position 4 of the fluorene. In anotherembodiment, SO₃H is at position, 1, 2, 3 or 4 of the fluorene or anycombination thereof.

In another embodiment, (PEG-S-MAL-Fmoc)n-OXM is represented by thefollowing structure:

In another embodiment, (PEG-S-MAL-FMS)n-OXM is represented by thefollowing structure:

In one embodiment, SO₃H is at position 2 of the fluorene. In anotherembodiment, SO₃H is at position 1 of the fluorene. In anotherembodiment, SO₃H is at position 3 of the fluorene. In anotherembodiment, SO₃H is at position 4 of the fluorene. In anotherembodiment, SO₃H is at position, 1, 2, 3 or 4 of the fluorene or anycombination thereof.

In another embodiment, pegylation of OXM includes reacting MAL-FMS-NHSor MAL-Fmoc-NHS with PEG-SH, thus forming a PEG-S-MAL-FMS-NHS orPEG-S-MAL-Fmoc-NHS conjugate, and then reacting it with the aminecomponent of OXM resulting in the desired (PEG-S-MAL-FMS)n-OXM or(PEG-S-MAL-Fmoc)n-OXM conjugate, respectively. In another embodiment,pegylation of peptides/proteins such as OXM are described in U.S. Pat.No. 7,585,837, which is incorporated herein by reference in itsentirety. In another embodiment, reverse-pegylation of peptides/proteinssuch as OXM with Fmoc or FMS are described in U.S. Pat. No. 7,585,837.

In another embodiment, PEG-S-MAL-Fmoc-NHS is represented by thefollowing structure

In another embodiment, PEG-S-MAL-FMS-NHS is represented by the followingstructure:

In one embodiment, SO₃H is at position 2 of the fluorene. In anotherembodiment, SO₃H is at position 1 of the fluorene. In anotherembodiment, SO₃H is at position 3 of the fluorene. In anotherembodiment, SO₃H is at position 4 of the fluorene. In anotherembodiment, SO₃H is at position, 1, 2, 3 or 4 of the fluorene or anycombination thereof.

In another embodiment, the phrases “long acting OXM” and “reversepegylated OXM” are used interchangeably and refer to a conjugate of thisinvention. In another embodiment, reverse pegylated OXM is composed ofPEG-FMS-OXM and PEG-Fmoc-OXM herein identified by the formulas:(PEG-FMS)n-OXM or (PEG-Fmoc)n-OXM, wherein n is an integer of at leastone, and OXM is linked to the FMS or Fmoc radical through at least oneamino group. In another embodiment, reverse pegylated OXM is composed ofPEG-S-MAL-FMS-OXM and PEG-S-MAL-Fmoc-OXM herein identified by theformulas: (PEG-S-MAL-FMS)n-OXM or (PEG-S-MAL-Fmoc)n-OXM, wherein n is aninteger of at least one, and OXM is linked to the FMS or Fmoc radicalthrough at least one amino group.

In one embodiment, this invention provides a process for preparing aPEG-S-MALFmoc-OXM or PEG-S-MALFMS-OXM wherein the amino terminal of saidOXM is linked to the Fmoc or FMS and wherein said OXM consists of theamino acid sequence set forth in SEQ ID NO: 1[His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-OH],

said process comprising reacting MAL-FMS-OXM or MAL-Fmoc-OXM:

withoxyntomodulin resin wherein the amino residues of said oxyntomodulin areprotected; to obtain MAL-Fmoc-protected OXM or MAL-FMS-protected OXM,wherein the amino residues of said oxyntomodulin are protected,respectively,followed by reaction with sulfhydryl PEG polymer (PEG-SH) whereinremoving said protecting groups and resin is conducted after or prior tosaid reaction with PEG-SH;to obtain PEG-S-MAL-Fmoc-OXM or PEG-S-MALFMS-OXM wherein the aminoterminal of said OXM is linked to the Fmoc or FMS.

In one embodiment, this invention provides a process for preparing aPEG-S-MAL-Fmoc-OXM or PEG-S-MALFMS-OXM conjugate, wherein said aminoresidue of Lys12 of said OXM is linked to said Fmoc or FMS and saidoxyntomodulin (OXM) consists of the amino acid sequence set forth in SEQID NO: 1[His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-OH],

said process comprising reacting MAL-FMS-OXM or MAL-Fmoc-OXM:

withoxyntomodulin resin wherein the amino residues (not including of Lys12)and the amino terminus of His¹ of said oxyntomodulin are protected;to obtain MAL-Fmoc-protected OXM or MAL-FMS-protected OXM, wherein theamino residues (not including of Lys12) and the amino terminus of His¹of said oxyntomodulin are protected, respectively;followed by reaction with sulfhydryl PEG polymer (PEG-SH) whereinremoving said protecting groups and said resin is conducted after orprior to the reaction with said PEG-SH; to yield PEG-S-MAL-Fmoc-OXM orPEG-S-MAL-FMS-OXM wherein said amino residue of Lys12 of said OXM islinked to said Fmoc or FMS.

In one embodiment, this invention provides a process for preparing aPEG-S-MAL-Fmoc-OXM or PEG-S-MALFMS-OXM conjugate, wherein said aminoresidue of Lys30 of said OXM is linked to said Fmoc or FMS and saidoxyntomodulin (OXM) consists of the amino acid sequence set forth in SEQID NO: 1[His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-OH],

said process comprising reacting MAL-FMS-OXM or MAL-Fmoc-OXM:

withoxyntomodulin resin wherein the amino residues (not including of Lys30)and the amino terminus of His¹ of said oxyntomodulin are protected;to obtain MAL-Fmoc-protected OXM or MAL-FMS-protected OXM, wherein theamino residues (not including of Lys30) and the amino terminus of His¹of said oxyntomodulin are protected, respectively;followed by reaction with sulfhydryl PEG polymer (PEG-SH) whereinremoving said protecting groups and said resin is conducted after orprior to the reaction with said PEG-SH; to yield PEG-S-MALFmoc-OXM orPEG-S-MALFMS-OXM wherein said amino residue of Lys12 of said OXM islinked to said Fmoc or FMS.

In another embodiment, the conjugation of PEG-S-MALFmoc or PEG-S-MALFMSto Lys12 or Lys30 or the amino terminus of OXM does not render the OXMinactive.

In one embodiment, the Lys12 variant is more effective at providingweight control than the other variants provided herein. In anotherembodiment, the Lys30 variant provided herein is more effective atachieving weight control than the other variants provided herein. Inanother embodiment, the amino variant provided herein is more effectiveat achieving to weight control than the other variants provided herein.

In one embodiment, the Lys12 variant is more effective at achievingchronic glycemic control than the other variants provided herein. Inanother embodiment, the Lys30 variant provided herein is more effectiveat achieving chronic glycemic control than the other variants providedherein. In another embodiment, the amino variant provided herein is moreeffective at achieving glycemic control than the other variants providedherein.

In additional embodiment the amino variant of PEG30-FMS-OXM is moreeffective at providing weight control than the other variants providedherein. In additional embodiment the amino variant of PEG30-FMS-OXM ismore effective at achieving glycemic control than the other variantsprovided herein. In another embodiment the amino variant ofPEG30-FMS-OXM is more effective at weight reduction than the othervariants provided herein. In another embodiment the amino variant ofPEG30-FMS-OXM is more effective at reduction of cumulative food intakethan the other variants provided herein. In another embodiment the aminovariant of PEG30-FMS-OXM is more effective at reduction of plasmaglucose intake than the other variants provided herein. In anotherembodiment the amino variant of PEG30-FMS-OXM is more effective atimproving glucose tolerance than the other variants provided herein. Inanother embodiment the amino variant of PEG30-FMS-OXM is more effectiveat reduction of terminal plasma cholesterol levels than the othervariants provided herein.

In one embodiment, PEG-S-MAL-Fmoc-OXM is effective at reduction ofterminal plasma fructosamine levels. In another embodiment, PEG-EMCS-OXMis effective at reduction of terminal plasma fructosamine levels. Inanother embodiment, the amino variant of PEG30-S-MAL-FMS-OXM iseffective at reduction of terminal plasma fructosamine levels. Inanother embodiment the amino variant of PEG30-S-MAL-FMS-OXM is moreeffective at reduction of terminal plasma fructosamine levels than theother variants provided herein.

Pharmaceutical Composition and Methods of Use

In one embodiment, this invention provides a pharmaceutical compositioncomprising the conjugate of this invention and a carrier and excipient.In another embodiment, the conjugate is represented by formula I-IV.

In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them are utilized for theprevention of hyperglycemia, for improving glycemic control, fortreatment of diabetes mellitus selected from the group consisting ofnon-insulin dependent diabetes mellitus (in one embodiment, Type 2diabetes), insulin-dependent diabetes mellitus (in one embodiment, Type1 diabetes), and gestational diabetes mellitus, or any combinationthereof. In another embodiment, conjugates of this invention andpharmaceutical compositions comprising them are utilized for treatingType 2 Diabetes. In another embodiment, the conjugates of this inventionand pharmaceutical compositions comprising them are utilized forincreasing sensitivity to insulin. In another embodiment, the conjugatesof this invention provided herein and pharmaceutical compositionscomprising them are utilized for reducing insulin resistance.

In another embodiment, the conjugates of this invention provided hereinand pharmaceutical compositions comprising them are utilized for thesuppression of appetite. In another embodiment, the conjugates of thisinvention provided herein and pharmaceutical compositions comprisingthem are utilized for inducing satiety. In another embodiment, theconjugates of this invention provided herein and pharmaceuticalcompositions comprising them are utilized for the reduction of bodyweight. In another embodiment, the conjugates of this invention providedherein and pharmaceutical compositions comprising them are utilized forthe reduction of body fat. In another embodiment, the conjugates of thisinvention provided herein and pharmaceutical compositions comprisingthem are utilized for the reduction of body mass index. In anotherembodiment, the conjugates of this invention provided herein andpharmaceutical compositions comprising them are utilized for thereduction of food consumption. In another embodiment, the conjugates ofthis invention provided herein and pharmaceutical compositionscomprising them are utilized for treating obesity. In anotherembodiment, the conjugates of this invention herein and pharmaceuticalcompositions comprising them are utilized for treating diabetes mellitusassociated with obesity. In another embodiment, the conjugates of thisinvention and pharmaceutical compositions comprising them are utilizedfor increasing heart rate. In another embodiment, the conjugates of thisinvention and pharmaceutical compositions comprising them are utilizedfor increasing the basal metabolic rate (BMR). In another embodiment,the conjugates of this invention and pharmaceutical compositionscomprising them are utilized for increasing energy expenditure. Inanother embodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them are utilized for inducing glucosetolerance. In another embodiment, the conjugates of this inventionprovided herein and pharmaceutical compositions comprising them areutilized for improving glycemic and lipid profiles. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them are utilized for inducing glycemic control.In one embodiment, glycemic control refers to non-high and/ornon-fluctuating blood glucose levels and/or non-high and/ornon-fluctuating glycosylated hemoglobin levels.

In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them are utilized for inhibitingweight increase, where in another embodiment, the weight increase is dueto fat increase. In another embodiment, the conjugates of this inventionherein and pharmaceutical compositions comprising them are utilized forreducing blood glucose levels. In another embodiment, the conjugates ofthis invention herein and pharmaceutical compositions comprising themare utilized for decreasing caloric intake. In another embodiment, theconjugates of this invention herein and pharmaceutical compositionscomprising them are utilized for decreasing appetite. In anotherembodiment, the conjugates of this invention herein and pharmaceuticalcompositions comprising them are utilized for weight control. In anotherembodiment, the conjugates of this invention provided herein andpharmaceutical compositions comprising them are utilized for inducing orpromoting weight loss. In another embodiment, the conjugates of thisinvention and pharmaceutical compositions comprising them are utilizedfor maintaining any one or more of a desired body weight, a desired BodyMass Index, a desired appearance and good health. In another embodiment,conjugates of this invention herein and pharmaceutical compositionscomprising them are utilized for controlling a lipid profile. In anotherembodiment, the conjugates of this invention herein and pharmaceuticalcompositions comprising them are utilized for reducing triglyceridelevels. In another embodiment, the conjugates of this invention hereinand pharmaceutical compositions comprising them are utilized forreducing glycerol levels. In another embodiment, the conjugates of thisinvention and pharmaceutical compositions comprising them are utilizedfor increasing adiponectin levels. In another embodiment, the conjugatesof this invention provided herein and pharmaceutical compositionscomprising them are utilized for reducing free fatty acid levels.

In one embodiment, the terms “reducing the level of” refers to areduction of about 1-10% relative to an original, wild-type, normal orcontrol level. In another embodiment, the reduction is of about 11-20%.In another embodiment, the reduction is of about 21-30%. In anotherembodiment, the reduction is of about 31-40%. In another embodiment, thereduction is of about 41-50%. In another embodiment, the reduction is ofabout 51-60%. In another embodiment, the reduction is of about 61-70%.In another embodiment, the reduction is of about 71-80%. In anotherembodiment, the reduction is of about 81-90%. In another embodiment, thereduction is of about 91-95%. In another embodiment, the reduction is ofabout 96-100%.

In one embodiment, the terms “increasing the level of” or “extending”refers to a increase of about 1-10% relative to an original, wild-type,normal or control level. In another embodiment, the increase is of about11-20%. In another embodiment, the increase is of about 21-30%. Inanother embodiment, the increase is of about 31-40%. In anotherembodiment, the increase is of about 41-50%. In another embodiment, theincrease is of about 51-60%. In another embodiment, the increase is ofabout 61-70%. In another embodiment, the increase is of about 71-80%. Inanother embodiment, the increase is of about 81-90%. In anotherembodiment, the increase is of about 91-95%. In another embodiment, theincrease is of about 96-100%.

In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them are utilized for reducingcholesterol levels. In one embodiment, the reduction in cholesterollevels is greater than the reduction observed after administration ofnative OXM. In one embodiment, the conjugates of this invention andpharmaceutical compositions comprising them lower cholesterol levels by60-70%. In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them lower cholesterol levels by50-100%. In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them lower cholesterol levels by25-90%. In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them lower cholesterol levels by50-80%. In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them lower cholesterol levels by40-90%. In another embodiment, the conjugates of this invention andpharmaceutical compositions comprising them are utilized for increasingHDL cholesterol levels.

In one embodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them may be used for the purposes describedherein without a significant decrease in effectiveness over the courseof administration. In one embodiment, conjugates of this invention andpharmaceutical compositions comprising them remains effective for 1 day.In another embodiment, conjugates of this invention and pharmaceuticalcompositions comprising them remains effective for 2-6 days. In oneembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remains effective for 1 week. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 2 weeks. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 3 weeks. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 4 weeks. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 6 weeks. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 2 months. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 4 months. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 6 months. In anotherembodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them remain effective for 1 year or more.

In one embodiment, the conjugates of this invention and pharmaceuticalcompositions comprising them may be used for the purposes describedherein and may be effective immediately upon administration of the firstdose. In another embodiment, conjugates of this invention andpharmaceutical compositions comprising them are effective after two ormore doses have been administered.

In another embodiment, methods of utilizing the conjugates of thisinvention and pharmaceutical compositions comprising them as describedhereinabove are applied to a human subject afflicted with a disease orcondition that can be alleviated, inhibited, and/or treated by OXM. Inanother embodiment, methods of utilizing the conjugates of thisinvention and pharmaceutical compositions comprising them as describedhereinabove are veterinary methods. In another embodiment, methods ofutilizing the conjugates of this invention and pharmaceuticalcompositions comprising them as described hereinabove are applied toanimals such as farm animals, pets, and lab animals. Thus, in oneembodiment, a subject of the present invention is feline, canine,bovine, porcine, murine, aquine, etc.

In another embodiment, the present invention provides a method oftreating or reducing a disease treatable or reducible by OXM or apharmaceutical formulation comprising the same, in a subject, comprisingthe step of administering to a subject a therapeutically effectiveamount of the conjugates of this invention, thereby treating or reducinga disease treatable or reducible by OXM in a subject.

In another embodiment, OXM, “peptide” or “protein” as used hereinencompasses native peptides (either degradation products, syntheticallysynthesized proteins or recombinant proteins) and peptidomimetics(typically, synthetically synthesized proteins), as well as peptoids andsemipeptoids which are protein analogs, which have, in some embodiments,modifications rendering the proteins even more stable while in a body ormore capable of penetrating into cells.

In another embodiment, a “PEG-Fmoc-OXM and/or a PEG-FMS-OXM variant” isa conjugate of this invention. In another embodiment, a “PEG-Fmoc-OXMand/or a PEG-FMS-OXM variant” refers to PEG-S-MAL-Fmoc-OXM orPEG-S-MAL-FMS-OXM respectively and is a conjugate of this invention. Inanother embodiment, a conjugate of this invention is represented byformula I-IV. In another embodiment, a conjugate of this invention is aPEG linked OXM via either FMS or Fmoc, wherein the OXM is linked toeither FMS or Fmoc via Lys12 of the OXM, or via Lys30 of the OXM or viathe amino terminus of the OXM. In another embodiment, the pharmaceuticalcomposition comprises OXM peptide of the present invention between 0.005to 0.1 milligrams/kg in an injectable solution. In another embodiment,the pharmaceutical composition comprises from 0.005 to 0.5 milligrams/kgOXM peptide. In another embodiment, the pharmaceutical compositioncomprises from 0.05 to 0.1 micrograms OXM peptide.

In another embodiment, pharmaceutical composition comprising a conjugateof this invention is administered once a day. In another embodiment, apharmaceutical composition comprising a conjugate of this invention isadministered once every 36 hours. In another embodiment, pharmaceuticalcomposition comprising a conjugate of this invention is administeredonce every 48 hours. In another embodiment, pharmaceutical compositioncomprising a conjugate of this invention is administered once every 60hours. In another embodiment, a pharmaceutical composition comprising aconjugate of this invention is administered once every 72 hours. Inanother embodiment, a pharmaceutical composition comprising a conjugateof this invention is administered once every 84 hours. In anotherembodiment, a pharmaceutical composition comprising a conjugate of thisinvention is administered once every 96 hours. In another embodiment, apharmaceutical composition comprising a conjugate of this invention isadministered once every 5 days. In another embodiment, a pharmaceuticalcomposition comprising a conjugate of this invention is administeredonce every 6 days. In another embodiment, a pharmaceutical compositioncomprising a conjugate of this invention is administered once every 7days. In another embodiment, a pharmaceutical composition comprising aconjugate of this invention is administered once every 8-10 days. Inanother embodiment, a pharmaceutical composition comprising a conjugateof this invention is administered once every 10-12 days. In anotherembodiment, a pharmaceutical composition comprising a conjugate of thisinvention is administered once every 12-15 days. In another embodiment,a pharmaceutical composition comprising a conjugate of this invention isadministered once every 15-25 days.

In another embodiment, a conjugate of this invention is administered byan intramuscular (IM) injection, subcutaneous (SC) injection, orintravenous (IV) injection once a week.

In another embodiment, the conjugate of this invention can be providedto the individual per se. In one embodiment, the reverse pegylated OXMof the present invention can be provided to the individual as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

In another embodiment, a “pharmaceutical composition” refers to apreparation of long-acting OXN as described herein with other chemicalcomponents such as physiologically suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism. In another embodiment, a reverse pegylatedOXM is accountable for the biological effect. In another embodiment, thepharmaceutical composition of this invention comprises a conjugate ofthis invention, a pharmaceutically acceptable carrier and excipients.

In another embodiment, any of the compositions of this invention willcomprise at least a reverse pegylated OXM. In one embodiment, thepresent invention provides combined preparations. In one embodiment, “acombined preparation” defines especially a “kit of parts” in the sensethat the combination partners as defined above can be dosedindependently or by use of different fixed combinations withdistinguished amounts of the combination partners i.e., simultaneously,concurrently, separately or sequentially. In some embodiments, the partsof the kit of parts can then, e.g., be administered simultaneously orchronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts. Theratio of the total amounts of the combination partners, in someembodiments, can be administered in the combined preparation. In oneembodiment, the combined preparation can be varied, e.g., in order tocope with the needs of a patient subpopulation to be treated or theneeds of the single patient which different needs can be due to aparticular disease, severity of a disease, age, sex, or body weight ascan be readily made by a person skilled in the art.

In another embodiment, the phrases “physiologically acceptable carrier”and “pharmaceutically acceptable carrier” which be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. In one embodiment, one of the ingredients includedin the pharmaceutically acceptable carrier can be for examplepolyethylene glycol (PEG), a biocompatible polymer with a wide range ofsolubility in both organic and aqueous media (Mutter et al. (1979).

In another embodiment, “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate administration of along-acting OXN. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In another embodiment, suitable routes of administration of the peptideof the present invention, for example, include oral, rectal,transmucosal, transnasal, intestinal or parenteral delivery, includingintramuscular, subcutaneous and intramedullary injections as well asintrathecal, direct intraventricular, intravenous, intraperitoneal,intranasal, or intraocular injections.

The present invention also includes reverse pegylated OXM for use in themanufacture of a medicament for administration by a route peripheral tothe brain for any of the methods of treatment described above. Examplesof peripheral routes include oral, rectal, parenteral e.g. intravenous,intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual,nasal, subcutaneous or transdermal administration, includingadministration by inhalation. Preferred dose amounts of OXM for themedicaments are given below.

The present invention provides a pharmaceutical composition comprisingreverse pegylated OXM and a pharmaceutically suitable carrier, in a formsuitable for oral, rectal, parenteral, e.g. intravenous, intramuscular,or intraperitoneal, mucosal e.g. buccal, sublingual, nasal, subcutaneousor transdermal administration, including administration by inhalation.If in unit dosage form, the dose per unit may be, for example, asdescribed below or as calculated on the basis of the per kg doses givenbelow.

In another embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body. In anotherembodiment, a reverse pegylated OXM is formulated in an intranasaldosage form. In another embodiment, a reverse pegylated OXM isformulated in an injectable dosage form.

Various embodiments of dosage ranges are contemplated by this invention:the OXM peptide component within of the reverse pegylated OXMcomposition is administered in a range of 0.01-0.5 milligrams/kg bodyweight per 3 days (only the weight of the OXM within the reversepegylated OXM composition is provided as the size of PEG can differsubstantially). In another embodiment, the OXM peptide component withinof the reverse pegylated OXM composition is administered in a range of0.01-0.5 milligrams/kg body weight per 7 days. In another embodiment,the OXM peptide component within of the reverse pegylated OXMcomposition is administered in a range of 0.01-0.5 milligrams/kg bodyweight per 10 days. In another embodiment, the OXM peptide componentwithin of the reverse pegylated OXM composition is administered in arange of 0.01-0.5 milligrams/kg body weight per 14 days. In anotherembodiment, unexpectedly, the effective amount of OXM in a reversepegylated OXM composition is ¼- 1/10 of the effective amount of freeOXM. In another embodiment, unexpectedly, reverse pegylation of OXMenables limiting the amount of OXM prescribed to a patient by at least50% compared with free OXM. In another embodiment, unexpectedly, reversepegylation of OXM enables limiting the amount of OXM prescribed to apatient by at least 70% compared with free OXM. In another embodiment,unexpectedly, reverse pegylation of OXM enables limiting the amount ofOXM prescribed to a patient by at least 75% compared with free OXM. Inanother embodiment, unexpectedly, reverse pegylation of OXM enableslimiting the amount of OXM prescribed to a patient by at least 80%compared with free OXM. In another embodiment, unexpectedly, reversepegylation of OXM enables limiting the amount of OXM prescribed to apatient by at least 85% compared with free OXM. In another embodiment,unexpectedly, reverse pegylation of OXM enables limiting the amount ofOXM prescribed to a patient by at least 90% compared with free OXM.

In another embodiment, the OXM peptide component within of the reversepegylated OXM composition is administered in a range of 0.01-0.5milligrams/kg body weight once every 3 days (only the weight of the OXMwithin the reverse pegylated OXM composition is provided as the size ofPEG can differ substantially). In another embodiment, the OXM peptidecomponent within of the reverse pegylated OXM composition isadministered in a range of 0.01-0.5 milligrams/kg body weight once every7 days. In another embodiment, the OXM peptide component within of thereverse pegylated OXM composition is administered in a range of 0.01-0.5milligrams/kg body weight once every 10 days. In another embodiment, theOXM peptide component within of the reverse pegylated OXM composition isadministered in a range of 0.01-0.5 milligrams/kg body weight once every14 days.

In another embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 2-fold and reducesthe effective weekly dose by at least 2-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 3-fold and reducesthe effective weekly dose by at least 3-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 4-fold and reducesthe effective weekly dose by at least 4-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 5-fold and reducesthe effective weekly dose by at least 5-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 6-fold and reducesthe effective weekly dose by at least 6-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, effective dosing frequency and effective weekly doseare based on: (1) the weight of administered OXM component within thereverse pegylated OXM composition; and (2) the weight of administeredOXM component within the free OXM (unmodified OXM) composition.

In another embodiment, the methods of the invention include increasingthe compliance of patients afflicted with chronic illnesses that are inneed of OXM therapy. In another embodiment, the methods of the inventionenable reduction in the dosing frequency of OXM by reverse pegylatingOXM as described hereinabove. In another embodiment, the methods of theinvention include increasing the compliance of patients in need of OXMtherapy by reducing the frequency of administration of OXM. In anotherembodiment, reduction in the frequency of administration of the OXM isachieved thanks to reverse pegylation which render the OXM more stableand more potent. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of increasing T½ ofthe OXM. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of reducing bloodclearance of OXM. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of increasing T1/2 ofthe OXM. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of increasing the AUCmeasure of the OXM.

In another embodiment, a reverse pegylated OXM is administered to asubject once a day. In another embodiment, a reverse pegylated OXM isadministered to a subject once every two days. In another embodiment, areverse pegylated OXM is administered to a subject once every threedays. In another embodiment, a reverse pegylated OXM is administered toa subject once every four days. In another embodiment, a reversepegylated OXM is administered to a subject once every five days. Inanother embodiment, a reverse pegylated OXM is administered to a subjectonce every six days. In another embodiment, a reverse pegylated OXM isadministered to a subject once every week. In another embodiment, areverse pegylated OXM is administered to a subject once every 7-14 days.In another embodiment, a reverse pegylated OXM is administered to asubject once every 10-20 days. In another embodiment, a reversepegylated OXM is administered to a subject once every 5-15 days. Inanother embodiment, a reverse pegylated OXM is administered to a subjectonce every 15-30 days.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of OXM of the invention, each of which is in oneembodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or inanother embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. Thepharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmellose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the long-acting OXN as knownto one skilled in the art.

In another embodiment, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in another embodimentare aqueous solutions or emulsions comprising a safe and effectiveamount of the compounds of the present invention and optionally, othercompounds, intended for topical intranasal administration. In someembodiments, the compositions comprise from about 0.001% to about 10.0%w/v of a subject compound, more preferably from about 00.1% to about2.0, which is used for systemic delivery of the compounds by theintranasal route.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, subcutaneous or intramuscular injectionof a liquid preparation. In another embodiment, liquid formulationsinclude solutions, suspensions, dispersions, emulsions, oils and thelike. In one embodiment, the pharmaceutical compositions areadministered intravenously, and are thus formulated in a form suitablefor intravenous administration. In another embodiment, thepharmaceutical compositions are administered intra-arterially, and arethus formulated in a form suitable for intra-arterial administration. Inanother embodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces, and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the compounds of the present invention arecombined with an additional appropriate therapeutic agent or agents,prepared and applied as solutions, suspensions, or emulsions in aphysiologically acceptable diluent with or without a pharmaceuticalcarrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, pharmaceutical compositions for use in accordancewith the present invention is formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of OXM into preparationswhich, can be used pharmaceutically. In one embodiment, formulation isdependent upon the route of administration chosen.

In one embodiment, injectables, of the invention are formulated inaqueous solutions. In one embodiment, injectables, of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In another embodiment, formulations for injection arepresented in unit dosage form, e.g., in ampoules or in multidosecontainers with optionally, an added preservative. In anotherembodiment, compositions are suspensions, solutions or emulsions in oilyor aqueous vehicles, and contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

The compositions also comprise, in another embodiment, preservatives,such as benzalkonium chloride and thimerosal and the like; chelatingagents, such as edetate sodium and others; buffers such as phosphate,citrate and acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In one embodiment, pharmaceutical compositions for parenteraladministration include aqueous solutions of the active preparation inwater-soluble form. Additionally, suspensions of long acting OXM, insome embodiments, are prepared as appropriate oily or water basedinjection suspensions. Suitable lipophilic solvents or vehicles include,in some embodiments, fatty oils such as sesame oil, or synthetic fattyacid esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions contain, in some embodiments, substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In another embodiment, the suspensionalso contain suitable stabilizers or agents which increase thesolubility of long acting OXM to allow for the preparation of highlyconcentrated solutions.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990).

In one embodiment, long acting OXM is in powder form for constitutionwith a suitable vehicle, e.g., sterile, pyrogen-free water basedsolution, before use. Compositions are formulated, in some embodiments,for atomization and inhalation administration. In another embodiment,compositions are contained in a container with attached atomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In one embodiment, pharmaceutical compositions suitable for use incontext of the present invention include compositions wherein longacting OXM is contained in an amount effective to achieve the intendedpurpose. In another embodiments, a therapeutically effective amountmeans an amount of long acting OXM effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

The compositions also comprise preservatives, such as benzalkoniumchloride and thimerosal and the like; chelating agents, such as edetatesodium and others; buffers such as phosphate, citrate and acetate;tonicity agents such as sodium chloride, potassium chloride, glycerin,mannitol and others; antioxidants such as ascorbic acid, acetylcystine,sodium metabisulfote and others; aromatic agents; viscosity adjustors,such as polymers, including cellulose and derivatives thereof; andpolyvinyl alcohol and acid and bases to adjust the pH of these aqueouscompositions as needed. The compositions also comprise local anestheticsor other actives. The compositions can be used as sprays, mists, drops,and the like.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. The choice of apharmaceutically-acceptable carrier to be used in conjunction with thecompound is basically determined by the way the compound is to beadministered. If the subject compound is to be injected, in oneembodiment, the pharmaceutically-acceptable carrier is sterile,physiological saline, with a blood-compatible suspending agent, the pHof which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hydroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts.) Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

In one embodiment, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Inanother embodiment, the modified compounds exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified compounds. In one embodiment, modificationsalso increase the compounds solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In another embodiment, the desired in vivo biological activityis achieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In another embodiment, preparation of effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the long actingOXM as described herein can be determined by standard pharmaceuticalprocedures in vitro, in cell cultures or experimental animals. In oneembodiment, the data obtained from these in vitro and cell cultureassays and animal studies can be used in formulating a range of dosagefor use in human. In one embodiment, the dosages vary depending upon thedosage form employed and the route of administration utilized. In oneembodiment, the exact formulation, route of administration and dosagecan be chosen by the individual physician in view of the patient'scondition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical carrier are also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

In another embodiment, a reverse pegylated OXM as described herein isadministered via systemic administration. In another embodiment, areverse pegylated OXM as described herein is administered byintravenous, intramuscular or subcutaneous injection. In anotherembodiment, a reverse pegylated OXM as described herein is lyophilized(i.e., freeze-dried) preparation in combination with complex organicexcipients and stabilizers such as nonionic surface active agents (i.e.,surfactants), various sugars, organic polyols and/or human serumalbumin. In another embodiment, a pharmaceutical composition comprises alyophilized reverse pegylated OXM as described in sterile water forinjection. In another embodiment, a pharmaceutical composition comprisesa lyophilized reverse pegylated OXM as described in sterile PBS forinjection. In another embodiment, a pharmaceutical composition comprisesa lyophilized reverse pegylated OXM as described in sterile 0.9% NaClfor injection.

In another embodiment, the pharmaceutical composition comprises areverse pegylated OXM as described herein and complex carriers such ashuman serum albumin, polyols, sugars, and anionic surface activestabilizing agents. See, for example, WO 89/10756 (Hara etal.—containing polyol and p-hydroxybenzoate). In another embodiment, thepharmaceutical composition comprises a reverse pegylated OXM asdescribed herein and lactobionic acid and an acetate/glycine buffer. Inanother embodiment, the pharmaceutical composition comprises a reversepegylated OXM as described herein and amino acids, such as arginine orglutamate that increase the solubility of interferon compositions inwater. In another embodiment, the pharmaceutical composition comprises alyophilized reverse pegylated OXM as described herein and glycine orhuman serum albumin (HSA), a buffer (e g. acetate) and an isotonic agent(e.g NaCl). In another embodiment, the pharmaceutical compositioncomprises a lyophilized reverse pegylated OXM as described herein andphosphate buffer, glycine and HSA.

In another embodiment, the pharmaceutical composition comprising apegylated or reverse pegylated OXM as described herein is stabilizedwhen placed in buffered solutions having a pH between about 4 and 7.2.In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein is stabilized with an aminoacid as a stabilizing agent and in some cases a salt (if the amino aciddoes not contain a charged side chain).

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein is a liquid compositioncomprising a stabilizing agent at between about 0.3% and 5% by weightwhich is an amino acid.

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein provides dosing accuracy andproduct safety. In another embodiment, the pharmaceutical compositioncomprising a reverse pegylated OXM as described herein provides abiologically active, stable liquid formulation for use in injectableapplications. In another embodiment, the pharmaceutical compositioncomprises a non-lyophilized reverse pegylated OXM as described herein.

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein provides a liquid formulationpermitting storage for a long period of time in a liquid statefacilitating storage and shipping prior to administration.

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein comprises solid lipids asmatrix material. In another embodiment, the injectable pharmaceuticalcomposition comprising a reverse pegylated OXM as described hereincomprises solid lipids as matrix material. In another embodiment, theproduction of lipid microparticles by spray congealing was described bySpeiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipidnanopellets for peroral administration (Speiser EP 0167825 (1990)). Inanother embodiment, lipids, which are used, are well tolerated by thebody (e. g. glycerides composed of fatty acids which are present in theemulsions for parenteral nutrition).

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein is in the form of liposomes(J. E. Diederichs and al., Pharm./nd. 56 (1994) 267-275).

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein comprises polymericmicroparticles. In another embodiment, the injectable pharmaceuticalcomposition comprising a reverse pegylated OXM as described hereincomprises polymeric microparticles. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises nanoparticles. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises liposomes. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises lipid emulsion. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises microspheres. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises lipid nanoparticles. In another embodiment,the pharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises lipid nanoparticles comprising amphiphiliclipids. In another embodiment, the pharmaceutical composition comprisinga reverse pegylated OXM as described herein comprises lipidnanoparticles comprising a drug, a lipid matrix and a surfactant. Inanother embodiment, the lipid matrix has a monoglyceride content whichis at least 50% w/w.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the long acting OXM. Inone embodiment, the pack, for example, comprise metal or plastic foil,such as a blister pack. In one embodiment, the pack or dispenser deviceis accompanied by instructions for administration. In one embodiment,the pack or dispenser is accommodated by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions or human orveterinary administration. Such notice, in one embodiment, is labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert.

In one embodiment, it will be appreciated that the reverse pegylated OXMof the present invention can be provided to the individual withadditional active agents to achieve an improved therapeutic effect ascompared to treatment with each agent by itself. In another embodiment,measures (e.g., dosing and selection of the complementary agent) aretaken to adverse side effects which are associated with combinationtherapies.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Example 1 Preparation of PEG30-S-MAL-FMS-OXM

Synthesis of OXM

The oxyntomodulin amino acid sequence is set forth in the followingpeptide sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO: 1)

The peptide was synthesized by the solid phase method employing theFmoc-strategy throughout the peptide chain assembly (Almac Sciences,Scotland).

The peptide sequence was assembled using the following steps:

1. Capping

The resin was capped using 0.5M acetic anhydride (Fluka) solution in DMF(Rathburn).

2. Deprotection

Fmoc-protecting group was removed from the growing peptide chain using20% v/v piperidine (Rathburn) solution in DMF (Rathburn).

3. Amino Acid Coupling

0.5M Amino acid (Novabiochem) solution in DMF (Rathbum) was activatedusing 1M HOBt (Carbosynth) solution in DMF (Rathbum) and 1M DIC(Carbosynth) solution in DMF (Rathbum). 4 equivalents of each amino acidwere used per coupling.

The crude peptide is cleaved from the resin and protecting groupsremoved by stirring in a cocktail of Triisopropylsilane (Fluka), water,dimethylsulphide (Aldrich), ammonium iodide (Aldrich) and TFA (AppliedBiosystems) for 4 hours. The crude peptide is collected by precipitationfrom cold diethyl ether.

Peptide Purification

Crude peptide was dissolved in acetonitrile (Rathburn)/water (MilliQ)(5:95) and loaded onto the preparative HPLC column. The chromatographicparameters are as follows:

Column: Phenomenex Luna C18 250 mm×30, 15 μm, 300 A

Mobile Phase A: water+0.1% v/v TFA (Applied Biosystems)

Mobile Phase B: acetonitrile (Rathburn)+0.1% v/v TFA (AppliedBiosystems)

UV Detection: 214 or 220 nm

Gradient: 25% B to 31% B over 4 column volumes

Flow rate 43 mL/min

Synthesis of MAL-FMS-NHS

The synthesis of compounds 2-5 is based on the procedures described byAlbericio et al. in Synthetic Communication, 2001, 31(2), 225-232.

2-(Boc-amino)fluorene (2)

2-Aminofluorene (18 g, 99 mmol) was suspended in a mixture ofdioxane:water (2:1) (200 ml) and 2N NaOH (60 ml) in an ice bath withmagnetic stirring. Boc₂O (109 mmol, 1.1 eq) was then added and stirringcontinued at RT. The reaction was monitored by TLC (Rf=0.5, Hex./EthylAcetate 2:1) and the pH maintained between 9-10 by addition of 2N NaOH.At reaction completion, the suspension was acidified with 1M KHSO4 topH=3. The solid was filtered and washed with cold water (50 ml),dioxane-water (2:1) and then azeotroped with toluene twice before usingit in the next step.

9-Formyl-2-(Boc-amino)fluorene (3)

In a 3 necked RBF, NaH (60% in oil; 330 mmol, 3.3 eq) was suspended indry THF (50 ml), a solution of -(Boc-amino)fluorine described in step 2(28 g; 100 mmol) in dry THF (230 ml) was added dropwise over 20 minutes.A thick yellow slurry was observed and the mixture stirred for 10minutes at RT under nitrogen. Ethyl formate (20.1 ml, 250 mmol, 2.5 eq)was added dropwise (Caution: gas evolution). The slurry turned to a palebrown solution. The solution was stirred for 20 minutes. The reactionwas monitored by TLC (Rf=0.5, Hex./Ethyl acetate 1:1) and when onlytraces of starting material was observed, it was quenched with icedwater (300 ml). The mixture was evaporated under reduce pressure untilmost of the THF has been removed. The resulting mixture was treated withacetic acid to pH=5. The white precipitate obtained was dissolved inethyl acetate and the organic layer separated. The aqueous layer wasextracted with ethyl acetate and all the organic layer combined andwashed with saturated sodium bicarbonate, brine and dried over MgSO₄.After filtration and solvent removal a yellow solid was obtained. Thismaterial was used in the next step.

9-Hydroxymethyl-2-(Boc-amino)fluorene (4)

Compound 3 from above was suspended in MeOH (200 ml) and sodiumborohydride was added portion wise over 15 minutes. The mixture wasstirred for 30 minutes (caution: exothermic reaction and gas evolution).The reaction was monitored by TLC (Rf=0.5, Hex./EtOAc 1:1) and wascompleted. Water (500 ml) was added and the pH adjusted to pH 5 withacetic acid. The work-up involved extraction twice with ethyl acetate,washing the combined organic layers with sodium bicarbonate and brine,drying over MgSO₄, filtration and concentration to dryness. The crudeproduct obtained was purified by flask chromatography usingHeptane/EtOAc (3:1) to give a yellow foam (36 g, 97.5% purity, traces ofethyl acetate and diethyl ether observed in the 1H-NMR).

MAL-Fmoc-NHS (7)

To a clean dry 500 ml RBF with overhead agitation was chargedtriphosgene (1.58 g, 0.35 eq.) in dry THF (55 ml) to form a solution atambient. This was cooled to 0° C. with an ice/water bath and a solutionof NHS (0.67 g, 0.38 eq) in dry THF (19 ml) added dropwise over 10minutes under nitrogen at 0° C. The resultant solution was stirred for30 minutes. A further portion of NHS (1.34 g, 0.77 eq) in dry THF (36ml) was added dropwise at 0° C. over 10 minutes and stirred for 15minutes.

Compound 6 (5.5 g, 1 eq), dry THF (55 ml) and pyridine (3.07 ml, 2.5 eq)were stirred together to form a suspension. This was added to the NHSsolution in portions a 0-5° C. and then allowed to go to RT by removingthe ice bath.

After 20 hours the reaction was stop (starting material still present,if the reaction is pushed to completion a dimmer impurity has beenobserved).

The reaction mixture was filtered and to the filtrate, 4% brine (200 ml)and EtOAc (200 ml) were added. After separation, the organic layer waswashed with 5% citric acid (220 ml) and water (220 ml). The organiclayer was then concentrated to give 7.67 g of MAL-Fmoc-NHS (purity is93-97%). The material was purified by column chromatography using agradient cyclohexane/EtOAc 70:30 to 40:60. The fractions containingproduct were concentrated under vacuum to give 3.47 g (45%) ofMAL-Fmoc-NHS.

MAL-FMS-NHS-(A)

To a solution of MAL-Fmoc-NHS (100 mg, 0.2 mmol) in trifluoroacetic acid(10 ml), chlorosulfonic acid (0.5 ml) was added. After 15 minutes,ice-cold diethyl ether (90 ml) was added and the product precipitated.The material was collected by centrifugation, washed with diethyl etherand dried under vacuum. 41.3 mg (35%) of beige solid was obtained.

MAL-FMS-NHS-(B)

Starting material Mal-Fmoc-NHS was dissolved in neat TFA (typically 520mL) under an inert atmosphere for typically 5 minutes. 6 eqchlorosulfonic acid were dissolved in neat TFA (typically 106 mL) andadded dropwise to the reaction mixture (typically 45 minutes). Aftercompletion of sulfonation (typically 50 minutes) the reaction mixturewas poured on cold diethyl ether (typically 25.4 L) for precipitation.Filtration of the precipitate and drying in vacuum (typically 90minutes) afforded Mal-FMS-NHS (purity is 93-97%), which was subjecteddirectly to the coupling stage. Mal-FMS-NHS was obtained in sufficientpurities between 93%-97%.

Example 1A Conjugation of OXM+PEGSH+MAL-FMS-NHS-(A)—“One Pot Reaction”,to Yield Heterogenous Conjugate of PEG30-S-MAL-FMS-OXM (MOD 6030)

Heterogeneous conjugation of the 3 amine sites in the OXM peptide(Lys12, Lys30 and amino terminal) performed as a “one pot reaction” inwhich 1 eq from each component: OXM, mPEG-SH and FMS linker was mixedtogether at pH 7.2 for 30 min. The reaction was stopped by adding aceticacid to reduce PH to 4.

Synthesis of the heterogeneous conjugate (MOD-6030, FIG. 1,PEG₃₀-FMS-OXM) was performed as follows: MAL-FMS-NHS-(A) [as describedabove] was mixed with OXM and PEG(30)-SH (as a one pot reaction). TheMAL-FMS-NHS-(A) spacer was coupled to OXM by its NHS activated ester onone side and by PEG-SH connected to the maleimide group on the otherside simultaneously. This way, a heterogeneous mixture ofPEG-S-MAL-FMS-OXM conjugate is composed of three variants connected byone of the 3 amines of the OXM peptide (N-terminal, Lys₁₂ and Lys₃₀).

In the heterogenous conjugation the oxyntomodulin synthesis is completedand all protection groups are removed during cleavage and therefore theones with primary amine can further react with the NHS group. CrudeOxyntomodulin is purified and a one pot reaction takes place.

Example 1B Conjugation of OXM+PEGSH+MAL-FMS-NHS-(A)—Two Step Process, toYield Homogeneous Conjugate of PEG30-S-MAL-FMS-OXM

The conjugation procedure was further developed into a two-step processin which attachment to the FMS spacer (MAL-FMS-NHS) was executed in acontrolled and site directed manner. In the first step, the FMS spacerwas coupled to the protected OXM* (on resin partially protected OXM withthe N-terminal OXM protected at the Lys12 and Lys30 as the preferredprotected OXM), then cleaved followed by de-protection and purificationof MAL-FMS-OXM (by RP-HPLC).

*During peptide synthesis of OXM using Fmoc-SPPS methodology the aminoacids were protected by various protection group for each R group ofamino acid, which is deprotected during cleavage from the resin by TFA.In order to synthesize the Lys12 or Lys 30 site directed coupling of theFMS, ivDde were used to protect the amine group of the Lysine, e.g. forOXM-Lys12-FMS, the NH2 in the R group of Lys12 was added protected byivDde which was selectively removed by weak acid conditions while theall other amino acid in which other protection group were used, werestill protected. For the specific N-terminal coupling, a routine SPPSwas used. i.e. the synthesis of OXM was completed followed by additionof MAL-FMS-NHS which was coupled only to the non-protected N-terminalgroup.

The second step was the attachment of PEG30-SH to the purifiedhomogeneous MAL-FMS-OXM. The final conjugated product(PEG30-S-MAL-FMS-OXM) is further purified by RP-HPLC. Additionalpurification steps may be applied such as Ion exchange or SEC-HPLC orany other purification step.

Three peptides on resin were synthesized using Fmoc solid phasestrategy. For synthesis of the homogeneous conjugate connected by aminoacid lysine at position 12 or 30 of the OXM, a selective protectinggroup was applied for either Lys12 or Lys30 of OXM as ivDde(1-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidine)ethyl]), which can beremoved under basic conditions while the rest of the peptide is still onthe resin with the other protective groups.

Therefore, three resin-bound OXMs were synthesized: N-terminal-usingprotection groups suitable for solid phase synthesis with Fmoc strategy(usually Boc protecting group is used for the ε amine) and Lys₁₂ orLys₃₀ with ivDde protection group. These OXM peptides were intended forfurther selective coupling with the FMS linker.

Homogenous conjugates performed as ‘on resin synthesis’. The conjugatesynthesized in two steps:

1. Coupling between the OXM and MAL-FMS-NHS, cleavage and purification.

2. Pegylation of MAL-FMS-OXM with PEG₃₀-SH. In this procedure, thecoupling of the MAL-FMS-NHS compound is done with any one of theprotected OXMs (free N-terminal-OXM, free Lys12-OXM or free Lys30-OXM),while it is bound to the resin. The protected OXM was protected at theother free amine sites, allowing the specific un-protected desired aminosite on OXM to react with the NHS moiety on MAL-FMS-NHS. The purifiedMAL-FMS-OXM was reacted with the PEG30-SH to produce crude conjugatewhich was purified using HPLC (RP or Cation exchange or both).Coupling MAL-FMS-NHS (A) to Lys12/Lys30 Protected N-Terminal OXM(MOD-6031):

MAL-FMS-NHS linker solution (0.746 ml, 10 mg/ml in DMF, 2 eq) was addedto Lys12/Lys30 protected N-terminal OXM resin* (1 eq, 200 mg resin,31.998 μmol/g free amine). DMF was added until resin was just freelymobile and then sonicated for 19 hrs. Resin was washed with DMF andMethanol before drying overnight in vacuum desiccator. The cleavagecocktail contained TFA/TIS/H2O. The cleavage was performed over 3.5 hrsat room temperature. After filtration of the resin, the MAL-FMS-OXM wasprecipitated in cold diethyl ether. 42.1 mg of crude MAL-FMS-OXM (36%pure) was obtained at the end of the cleavage stage.

Coupling MAL-FMS-NHS (A) to Lys₁₂ Site Directed OXM:

MAL-FMS-NHS linker solution (10 mg/ml in DMF, 2.5 equiv.) was added to(Lys12)OXM resin (1 equiv.) with addition of DIEA (5 equiv.). DMF wasadded until resin was just freely mobile and then sonicated overnight.Resin was washed with DMF and Methanol before drying overnight in vacuumdesiccator. Cleavage and precipitation as described for N-terminal sitedirected.

Coupling MAL-FMS-NHS(A) to Lys₃₀ Site Directed OXM:

MAL-FMS-NHS linker (2.5 equiv.) was solubilized in DCM with addition ofDIEA (5 equiv.). This linker/DIEA solution was added to (Lys30)OXM resinthen sonicated overnight. Resin was washed with DCM and Methanol beforedrying overnight in vacuum desiccator. Cleavage and precipitation asdescribed for N-terminal site directed.

Purification

The resultant crude MAL-FMS-OXM from any of the resultant homogeneousintermediates produced above were purified in one portion under thefollowing conditions.

Sample diluent: 10% Acetonitrile in water

Column: Luna C18 (2), 100 Å, 250×21.2 mm

Injection flow rate: 9 ml/min

Run flow rate: 9 ml/min

Buffer A: Water (0.1% TFA)

Buffer B: Acetonitrile (0.1% TFA)

Gradient: 10-45% B over 32 mins

Monitoring: 230 nm

Any one of the homogeneous intermediates produced above were used toform a homogeneous conjugate in the following step:

Conjugation of PEG30SH to MAL-FMS-OXM

MAL-FMS-OXM solution (1 equiv, 15.1 mg in 1.5 ml DMF) was prepared.PEG30SH (1 equiv, 9.2 ml of 10 mg/ml in pH 6.5 phosphate buffer) wasadded to the MAL-FMS-OXM solution. The reaction mixture was then stirredfor 30 mins at room temperature before adding glacial acetic acid (200μl) to quench reaction by lowering the pH.

The resultant product was then purified using RP-HPLC to provide thedesired homogenous conjugate PEG-S-MAL-FMS-OXM (PEG-FMS-OXM).

Column: Luna C18 (2), 100 Å, 250×21.2 mm

Injection flow rate: 5 ml/min

Run flow rate: 20 ml/min

Buffer A: Water & 0.1% TFA

Buffer B: Acetonitrile/Water (75:25) & 0.1% TFA

Gradient: 10-65% B over 41 mins

Monitoring: 220, 240, 280 nm

Example 1C Conjugation of OXM+PEGSH+MAL-FMS-NHS-(B)—Two Step Process, toYield Homogeneous Conjugate of PEG30-S-MAL-FMS-OXM

Coupling was performed by suspending the OXM resin (typically 236 g in 2L DMF (Using the protected Lys12/Lys30 N-terminal OXM, or the protectedLys12/N-terminal OXM or the protected Lys30/N-terminal OXM*) in asolution of the MAL-FMS-NHS (B), in neat DMF/DCM (1:1, v/v, typicallyconcentration of 12 g/L) under an inert atmosphere, subsequentlyadjusting the reaction mixture to apparent pH of 6.0-6.5 with neat DIPEA(typically 7.5 mL). Coupling was carried out at RT with stirring. TheMal-FMS-NHS linker was added in two portions (first portion: 1.5 eq;second portion 0.5 eq Mal-FMS-NHS; eq calculated with respect to theloading of the peptide resin; second portion was added after drawing offthe first portion). Each coupling step was conducted between 22 and 24h. The following filtration, successive washing of the resin with DMF(typically 8.5 mL/g resin, 3 times), MeOH (typically 8.5 mL/g resin, 3times) and isopropyl ether (typically 8.5 mL/g resin, 3 times) andsubsequent drying in vacuum (between 69 and 118 h) afforded fullyprotected MAL-FMS-OXM resin. Typically amounts of 116 g up to 243 g ofMAL-FMS-OXM resin were obtained.

*During peptide synthesis of OXM using Fmoc-SPPS methodology the aminoacids were protected by various protection group for each R group ofamino acid, which is deprotected during cleavage from the resin by TFA.In order to synthesize the Lys12 or Lys 30 site directed coupling of theFMS, ivDde were used to protect the amine group of the Lysine, e.g. forOXM-Lys12-FMS, the NH2 in the R group of Lys12 was added protected byivDde which was selectively removed by weak acid conditions while theall other amino acid in which other protection group were used, werestill protected. For the specific N-terminal coupling, a routine SPPSwas used. i.e. the synthesis of OXM was completed followed by additionof MAL-FMS-NHS which was coupled only to the non-protected N-terminalgroup.

Cleavage:

Crude MAL-FMS-OXM was obtained by treatment of the peptide resin withTFA/H₂O/TIPS (84:8.5:7.5, v/v/v) for 3.5 h at RT. After 3.5 h 1 eqammonium iodide was added as solid for the Met(O)-reduction. After 4.0 hascorbic acid (1.5 eq) was added as a solid. The cleavage cocktail wasstirred for another 5 minutes and precipitated in isopropyl ether (IPE)(typically 5 mL per mL of cleavage cocktail). Isolation was performed byfiltration and drying in vacuum (typically between 41 and 90 h).

Purification

Two dimensional purification scheme were applied (instead of one)

The stationary phase and gradient were changed.

Sample diluent: 50% acetic acid

Column: Luna C8 (10 μm, 100 Å), 30 cm×25 cm

Injection flow rate: 1500 ml/min

Run flow rate: 1500 ml/min

Buffer system and gradient: 0.1% H3PO4 (pH 2) (A: 3%, B: 60% ACN)(gradient profile: 0% B—70 min—100% B) for the first dimension and 0.1%TFA eluent (pH 2) (A: 3%, B: 100% ACN) (gradient profile: 0% B—97min—100% B) for the second dimension.

Detected wavelength: 220 nm

Conjugation of PEGSH to MAL-FMS-OXM

The peptide MAL-FMS-OXM (B) (12.3 g, 1 eq) and PEG30-SH (1.1 eq., 67.8 g(active SH-groups)) were dissolved separately in 20 mM NaOAc buffer (pH4.7) containing 10% ACN (12 g/L for peptide and 10 g/L for PEG30-SH).After adjusting pH to 6.1 (by using aq. NaOAc, pH 9.3) the solution wasstirred under an inert atmosphere at RT for typically 1 h. Then, pH wasadjusted to 4.5-5.0 with AcOH (25% v/v) and the obtained reactionmixture was applied for preparative HPLC purification.

Sample diluent: crude from PEGylation reaction

Column: Luna C18(2) (10 μm, 100 Å), 20 cm×28 cm

Injection flow rate: 907 ml/min

Run flow rate: 907 ml/min

Buffer system: 0.1% TFA eluent (pH 2.0) (A: 5% ACN, B: 90% ACN)

Gradient profile: 5% B—30 min—5% B—66 min—78% B—1 min—90% B—15 min—90%

B

Detected wavelength: 220 nm

Purified fraction were pooled and lyophilized

Example 2 In-Vitro Characterization of GLP-1 Receptor Activation

In-Vitro Characterization of GLP-1 Receptors Activation

Activation of GLP-1 receptor was assessed using two different celllines; HTS163C2 (Millipore) and cAMP Hunter™ CHO-K1 GLP1R (Discoverx),both are over expressing the GLP-1 receptor. The HTS163C2 (Millipore)were seeded in 96 wells half-area white plate (Greiner) at a density of100,000 cells/nil and incubated for 24 hours at 37° C. The cells wereincubated with escalating concentrations of heterogeneous PEG30-FMS-OXMand 3 homogeneous PEG30-FMS-OXM variants (amino, Lys12 and Lys30). CellscAMP concentrations were quantified by HTRF assay (Cisbio 62AM4PEB) andEC50 parameter was analyzed by PRISM software. The cAMP Hunter™ CHO-K1GLP1R secretes cAMP upon binding of the ligand to the receptor. Cells ata density of 500000 cells/nil were seeded in 96 wells plate, and wereincubated for 24 h at 37° C. with 5% CO₂. Ligands were diluted indiluent contains IBMX and were added in duplicate to the culture wellsfor 30 min at 37° C. with 5% CO₂. The concentration range ofPEG30-FMS-OXM was 1.5*10⁻¹⁰ to 1.2*10⁻⁶ M. Lysis buffer and detectorreagents were added to the wells and cAMP concentrations were detectedusing a chemiluminescent signal. The dose dependent curves wereestablished and the binding affinities (EC50) of various ligands werecalculated using PRISM software by applying the best fit dose responsemodel (Four parameters).

GLP-1 receptor binding activation of PEG-S-MAL-FMS-OXM (MOD-6030;heterogeneous) and 3 different homogeneous variants ofPEG-S-MAL-FMS-OXM; the amino (MOD-6031), Lys12 and Lys30 were assessedusing two different cell-lines over expressing GLP-1 receptor; theMillipore HTS163C2 cell line and the cAMP Hunter™ CHO-K1 GLP1R. Thepotencies were determined by calculating the EC50 of each variant,followed by calculating the relative potency of each variant to theheterogeneous (MOD-6030) version (dividing EC50 of each homogenousvariant by the EC50 of the heterogeneous version and multiplying it by100). The EC50 values and calculated relative potencies are presented intable 4. For comparison, the binding affinity of OXM and GLP-1 to GLP-1receptor of cAMP Hunter CHO-K1 GLP1R cell line were measured.

TABLE 4 GLP-1 and Glucagon receptors binding activation Millipore cAMPHunter ™ cAMP Hunter ™ HTS163C2 CHO-K1 GLP1R CHO-K1 GCGR RelativeRelative Relative potency to potency to potency to EC50 heterogeneousheterogeneous heterogeneous (nM) (%) EC50 (nM) (%) EC50 (nM) (%) HeteroPEG₃₀- 76.2 100 8.14 ± 1.35 100 11.32 ± 3.26  100 FMS-OXM PEG₃₀-FMS-55.2 72.24 8.07 ± 0.21 99.1 10.31 ± 2.87  91.1 OXM AMINO PEG₃₀-FMS- 179234.9 9.42 ± 1.77 115.7 20.21 ± 4.12  178.5 OXM Lys₁₂ PEG₃₀-FMS- 307402.9 17.34 ± 2.37  213.0 6.12 ± 1.75 54.1 OXM Lys₃₀ Oxyntomodulin 1.38± 0.68 1.02 ± 0.32 (OXM) GLP-1 0.016 ± 0.006 NA Glucagon NA  0.04 ±0.011

The relative potencies of the homogeneous variants were compared to theheterogeneous version and summarized in Table 4. Comparable bioactivityof the amino variant and the heterogeneous variant exhibited a relativepotency of 72.2% and 99.1% measured using the Millipore HTS163C2 and thecAMP Hunter™ CHO-K1 GLP1R, respectively.

The Lys12 and Lys30 variants had shown 2 and 4 fold reduction of GLP-1receptor binding activation using the Millipore HTS163C2 cell line whileonly showing minor and a 2 fold reduction, respectively, using the cAMPHunter™ CHO-K1 GLP1R cell line. The fact the amino variant demonstratedsuperior binding activity compared to the other variants is unexpectedas the N-terminus of OXM was reported to be involved in the binding ofOXM to the GLP-1 receptor (Druce et al., 2008). Overall, comparablebioactivity was shown for the amino variant and the heterogeneousvariant. GLP-1 receptor binding activations of OXM and GLP-1 peptideswere measured. It was found that OXM and GLP-1 had shown higher receptorbinding activation by 5.9 and 508.7 fold compared to the heterogeneousPEG30-FMS-OXM.

Example 3 In-Vitro Characterization of Glucagon Receptor Activation

In-Vitro Characterization of Glucagon Receptors Activation

Activation of glucagon receptor was assessed using cAMP Hunter™ CHO-K1GCGR cell-line that over expresses glucagon-receptor. This cell-linesecretes cAMP upon binding of the ligand to the glucagon receptor. Cellswere seeded at a density of 500000 cells/ml in 96 wells plate, and wereincubated for 24 h at 37° C. with 5% CO₂. Ligands were diluted indiluent contains IBMX and were added in duplicate to the culture wellsfor 30 min at 37° C. with 5% CO₂. The concentration range of MOD-6031was 5.8*10⁻¹¹ to 2.7*10⁻⁷ M. Lysis buffer and detector reagents wereadded to the wells and cAMP concentrations were detected using achemiluminescent signal. The dose dependent curves were established andthe binding affinities (EC50) of various ligands were calculated usingPRISM software by applying the best fit dose response model (Fourparameters).

Binding affinities of PEG-S-MAL-FMS-OXM variants to the glucagonreceptor were determined using cAMP Hunter™ CHO-K1 GCGR cell-line thatover expresses glucagon-receptor. This cell line was used tocharacterize the heterogeneous PEG-S-MAL-FMS-OXM (MOD-6030) and 3different homogeneous variants of PEG-S-MAL-FMS-OXM; the amino(MOD-6031), Lys12 and Lys30. The potencies were determined bycalculating the EC50 of each variant, followed by calculating therelative potency of each variant to the heterogeneous version (dividingEC50 of each homogenous variant by the EC50 of the heterogeneous versionand multiplying the value by 100). The EC50 values and calculatedrelative potencies are presented in table 4 Amino variant showedcomparable binding activity to the heterogeneous version. The Lys30variant showed the highest bioactivity and Lys12 had shown 1.8 foldreductions. Glucagon receptor binding activations of OXM and glucagonpeptides were measured. It was found that OXM and glucagon had shownhigher receptor binding activation by 11.1 and 283 fold compared to theheterogeneous PEG30-S-MAL-FMS-OXM.

Example 4 Induction of Glucose Tolerance by PEG30-FMS-OXM Variants

C57BL/6 male mice were fasted overnight then weighed, and blood glucoselevels were measured by tail vein sampling using a handheld glucometer.Mice were IP injected with PEG-SH (vehicle), PEG30-FMS-OXM(Heterogeneous) and the three homogeneous variants of PEG30-FMS-OXM(amino, Lys12 and Lys30). Glucose (1.5 gr/kg) was administrated IP 15min after test article administration. Blood glucose levels weremeasured by tail vein sampling at prior to glucose administration and10, 20, 30, 60, 90, 120 and 180 min after glucose administration using ahandheld glucometer

In order to evaluate the in vivo activity of the heterogeneousPEG₃₀-S-MAL-FMS-OXM and the three PEG₃₀-S-MAL-FMS-OXM variants (amino,Lys₁₂ and Lys₃₀), the IPGTT model was applied. Overnight fasted C57BL/6mice were injected IP with the different compounds and a vehicle(PEG-SH) followed by IP injection of glucose and measurement of bloodglucose levels from the tail vein using a glucometer. PEG-SH (238.10nmol/kg), heterogeneous and homogeneous PEG₃₀-S-MAL-FMS-OXM, 100 nmol/kgpeptide content) were administered IP 15 min prior to glucose IPinjection (1.5 gr/kg). All the compounds induced glucose tolerancecompared to vehicle group. Surprisingly, the homogeneous amino variantwas slightly less potent compared to the two other variants and to theheterogeneous PEG₃₀-S-MAL-FMS-OXM (table 5, FIG. 3) reflected by theslightly higher glucose AUC compared to other variants, as opposed tothe in-vitro activity results. Yet, all variants significantly improvedglucose tolerance as compared to the vehicle PEG-SH control.

TABLE 5 Glucose tolerance in C57BL/6 mice AUC % AUC % AUC (-60- from AUCfrom 180) control (0-180) control PEG-SH 26857 100 22522 100Heterogeneous 18200 67.8 13541 60.1 PEG₃₀-S-MAL- FMS-OXM PEG30-S-MAL-19891 74.1 15781 70.1 FMS-OXM AMINO variant PEG30-S-MAL- 17652 65.713953 62.0 FMS-OXM Lys12 variant PEG30-S-MAL- 17818 66.3 13159 58.4FMS-OXM Lys30 variant

The heterogeneous and homogeneous variants of the reversiblePEG₃₀-S-MAL-FMS-OXM were shown to be active both in-vitro and in theIPGTT model in-vivo. Surprisingly, the in-vitro results were not alignedwith what is suggested in the literature, that the N-terminus of nativeOXM is involved in the peptide binding to the GLP-1 receptor; therefore,it was expected that the amino terminus variant would show the lowestpotency both in-vitro and in-vivo. However, the homogeneous aminovariant of PEG₃₀-S-MAL-FMS-OXM demonstrated improved GLP-1 receptoractivation compared to the two other homogeneous variants using twodifferent cell lines (table 4) while demonstrating comparable efficacyin the IPGTT in vivo model. The IPGTT in vivo model seems to presentcomparable activity (considering the variability between the animals).Although different in-vitro binding activates to the GLP-1R and the GCGRwere observed between the different PEG30-FMS-OXM variants, comparableability to induce glucose tolerance was shown (table 4 and 5).Unexpectedly, the superior in vitro activity of homogeneous aminoPEG₃₀-S-MAL-FMS-OXM as shown in the cAMP induction assay was notreflected in the in vivo IP glucose tolerance test. The homogeneousamino variants PEG₃₀-S-MAL-FMS-OXM showed the lowest glucose toleranceprofile compared to the two other variants and to the heterogeneousPEG₃₀-S-MAL-FMS-OXM. However, it still showed significant glucosetolerance effect in comparison to the vehicle (FIG. 3).

Example 5 Improvement of Body Weight, Glycemic and Lipid Profiles byPEG30-S-MAL-FMS-OXM Variants in Ob/Ob Mouse Model

Materials and Methods

Study 1:

Twenty five male ob/ob mice (male, B6. V-Lep¹⁵⁸ ob/OlaHsd, 5-6 weeks ofage, Harlan) were acclimatized to the facility (10 days) followed byhandling protocol whereby animals were handled as if to be dosed butwere actually not weighed or dosed (10 days). Subsequently, animalsunderwent baseline period for 7 days in which they were dosed twice aweek with the appropriate vehicle by the subcutaneous route in volume of20 ml/kg. Body weight, food and water intake were recorded daily, andsamples were taken for non-fasting and fasting glucose measurements andnon-fasting and fasting insulin measurements. Animals were subsequentlyallocated into five treatment groups (N=5) based on body weight andglycemic profile. Animals were dosed every four days (days: 1, 5, 9, 13and 16) as described in table 1. During the treatment period, foodintake, water intake and body weight have been measured and recordeddaily, before dosing. Several procedures and sampling have beenperformed: non-fasting and fasting glucose on days 2, 6, 14 and 17 (onday 17 only non-fasting glucose was measured), fasting and non-fastinginsulin (days 2, 6 and 14). Terminal samples on day 19 were analyzed forcholesterol.

TABLE 1 Study design Group Treatment (sc) Frequency n 1 PEG-SH (142.86mg/ml) Days 1, 5, 9, 13 and 16 5 2 PEGS-MAL-FMS-OXM Hetero Days 1, 5, 9,13 and 16 5 (MOD-6030). 2000 nmol/kg 3 Amino PEG-S-MAL-FMS-OXM Days 1,5, 9, 13 and 16 5 2000 nmol/kg 4 Lys12 PEG-S-MAL-FMS-OXM Days 1, 5, 9,13 and 16 5 2000 nmol/kg 5 Lys30 PEG-S-MAL-FMS-OXM Days 1, 5, 9, 13 and16 5 2000 nmol/kg

Study 2:

One hundred male ob/ob mice (5-6 weeks of age, Charles River) wereacclimatized to the facility (3 days) followed by handling protocolwhereby animals were handled as if to be dosed but were actually notweighed or dosed (7 days). Subsequently, animals were underwent baselineperiod for 7 days in which they were dosed twice a week with PEG30-SHvehicle (146 mg/ml) by a subcutaneous route in volume of 20 ml/kg. Bodyweight, food and water intake were recorded daily. Subsequently animalswere allocated into 8 treatment, control and pair fed groups (groupsA-H, N=8) (table 2). The pair fed group was pair-fed to the high dose(6000 nmol/kg) group of MOD-6031 and it was given the daily food rationequal to that eaten by its paired counterpart in group D the previousday. 3 additional groups (groups I-K, N=12) were administered withMOD-6031 at 1000, 3000 and 6000 nmol/kg and were used for sampling forPK analysis. PEG-SH vehicle (292 mg/ml), MOD-6031 at 1000, 3000 and 6000nmol/kg, and the pair fed groups were administered twice a week for 32days while OXM, Liraglutide® and PBS were administered bid. Body weight,food and water intake were measured daily. Non-fasting and fastingglucose were measured once a week, OGTT were performed on days 2 and 30.Terminal blood samples (day 33) were analyzed for glucose, insulin,Cholesterol, and MOD-6031, PEG-S-MAL-FMS-NHS and OXM concentrations.Mice in the PK groups received a single dose of MOD-6031 and bloodsamples were taken at 4, 8, 24, 36, 48, 72, 96 and 120 h (n=3 per timepoint) for PK analysis allows to quantify MOD-6031 and its compoundsconcentrations by LC-MS/MS method.

TABLE 2 Study design Group Treatment (sc) n Frequency A PEG30-SH Vehicle8 Twice a week on (292 mg/kg; 20 ml/kg) days 1, 4, 8, 11, 15, B MOD-60311000 nmoles/kg 8 18, 22, 25, 29 and C MOD-6031 3000 nmoles/kg 8 32 DMOD-6031 6000 nmoles/kg 8 E PEG30-SH Vehicle 8 (292 mg/kg) Pair-Fed toGroup D F PBS bid (10 ml/kg) 8 b.i.d for 32 days G OXM 6000 nmoles/kgbid (10 ml/kg) 8 H Liraglutide 0.1 mg/kg bid (10 ml/kg) 8 I MOD-60311000 nmoles/kg PK group 12 Single injection on J MOD-6031 3000 nmoles/kgPK group 12 day 1 K MOD-6031 6000 nmoles/kg PK group 12

Study 3:

Forty-two male ob/ob mice (7 weeks of age, Charles River, Italy) wereacclimatized to the facility (10 days) followed by handling protocolwhereby animals were handled as if to be dosed but were actually notweighed or dosed. Subsequently, animals underwent baseline period for 1week in which each animal have been dosed twice by the subcutaneousroute with PEG30-SH in volume of 20 ml/kg. Body weight, food and waterintake were recorded daily, and samples were taken for non-fasting andfasting glucose measurements and non-fasting and fasting insulinmeasurements. Animals were subsequently allocated into three treatment,control and pair-fed groups (group A, N=10, groups B-E, N=8) based onplasma glucose, body weight and daily food and water intake. The pairfed group was pair-fed to group B (PEG-S-MAL-FMOC-OXM) but was treatedwith PEG-SH (204.5 mg/kg). It was given the daily food ration equal tothat eaten by its paired counterpart in group B the previous day. Assuch, animals in Group E will be one day out of phase with Group B inall study procedures and measurements. During the study, animals weredosed every four days (days: 1, 5, 9, 13, 17, 21, 25 and 29) asdescribes in table 3. During the treatment period, food intake, waterintake and body weight have been measured and recorded daily, beforedosing. Several procedures and sampling have been performed: non-fastingglucose on days 1, 6, 14, 22 and 29, fasting glucose on days 10, 18 and26. On days 2 and 30 fasting glucose samples have been taken as part ofan OGTT procedure, in which insulin was measured in parallel to glucose.Terminal samples on day 33 were analyzed for cholesterol, triglyceridesand fructosamine.

TABLE 3 Study design Group Treatment (sc) Frequency N A PEG30-SH VehicleDays 1, 5, 9, 13 and 16 10 (204.5 mg/kg; 20 ml/kg) B PEG-S-MAL-FMOC-OXMDays 1, 5, 9, 13 and 16 8 (6000 nmoles/kg) C MOD-6031 (6000 nmoles/kg)Days 1, 5, 9, 13 and 16 8 D PEG-EMCS-OXM (6000 nmoles/kg) Days 1, 5, 9,13 and 16 8 E PEG30-SH Vehicle (204.5 mg/kg) Days 1, 5, 9, 13 and 16 8Pair-Fed to Group BResults

The ob/ob mouse model exhibits a mutation of the ob gene such that theycannot produce leptin and develop a phenotype characterized byhyperinsulinemia, obesity, hyperphagia, insulin resistance andsubsequently hyperglycaemia. These mice were used as a genetic model ofdiabetes in two different studies in order to evaluate the efficacy ofPEG30-FMS-OXM (Heterogeneous) and the three homogeneous variants ofPEG30-S-MAL-FMS-OXM (amino, Lys12 and Lys30).

Study 1: This study compared the efficacy of homogeneous variants(amino, Lys12 and Lys30) and the heterogeneous MOD-6030 whenadministered at 2000 nmol/kg. Reductions of body weight were obtainedfor all tested articles compared to vehicle (PEG-SH) group with finalreduction (on day 18) of 3.1%, 4.7%, 4.9% and 6.5% for Lys12, MOD-6030,amino and Lys30 variants, respectively (FIG. 4). Body weight reductionswere observed following drug injection on days 1, 5, 13 and 16 (FIG. 4).Reduction of food intake was observed for all treated groups followingdrug administration (except day 9) (FIG. 5). Measurement of glycemicparameters along the study had shown improvement of non-fasting glucose(FIG. 6A) for amino and Lys12 treated groups and improvement of fastingglucose for all treated groups (FIG. 6B). All treated groups showedsignificantly lower level of insulin compared to the control. Of note,the administered dose in this study was 2000 nmol/kg which is the lowereffective dose of MOD-6030 and thus the improvement of body weight, foodintake and glycemic profile were relatively moderate. Unexpectedly theamino variant was the only variant which showed superior efficacies inthe ability to reduce weight, inhibit food intake and to improveglycemic control. From a manufacturing perspective, on resin synthesisof the amino variant is the most straight forward procedure consideringthat the peptide in solid phase synthesis is extended from the aminoterminus. The terminal amine has preferred availability for couplingthan the internal amine groups of the Lysine at positions 12 and 30.This accessibility is reflected in the higher manufacturing yields ofthe amino variant as compared to the Lys12 and Lys30 variants. Anadditional benefit is that the synthesis towards the amino variantremains unchanged relative to OXM synthesis for the heterogeneousvariant, while the synthesis of Lys12 and Lys30 variants was modified bychanging the Lys used for the peptide synthesis and by the addition of aselective cleavage step (selectively removing the protecting group ofthe Lys). The OXM synthesis as previously developed for theheterogeneous was already optimized to achieve better yield androbustness. Overall, from a manufacturing perspective, synthesis ofamino variant on-resin is straight forward and possesses an advantageover the alternative variants. Being a homogenous variant, it also hasan advantage over a heterogeneous variant in that it is more suitablefor drug development and drug treatment.

Study 2: This study investigated the chronic effect of twice a weekadministration of MOD-6031 (the amino variants) at 1000, 3000 and 6000nmol/kg, on pharmacological and pharmacokinetic parameters in ob/obmouse model, while OXM and liraglutide (long-acting GLP-1 receptoragonist) were evaluated as reference compounds. The measuredpharmacological parameters were body weight, food and water intake,glucose control and lipid profile. Twice a week administration of highdose of MOD-6031 (6000 nmol/kg) significantly reduced food intake andbody weight (FIG. 7; FIG. 8), while the lower doses (3000 and 1000nmol/kg) had shown lower effects. At the conclusion of the study (day33) animals of 1000, 3000 and 6000 nmol/kg had shown body weightreduction of 5.2%, 12.3% and 28.3%, respectively. The pair fed group,which were paired to the high dose group and ate equal amount of food(except the fasting days), had a body weight reduction of 12.7% whileundergoing similar food intake. This phenomenon can be attributed to theability of the amino variant of PEG30-FMS-OXM to increase energyexpenditure and thus animals that were treated with 6000 nmol/kg of theamino variant had an increased reduction of body weight over the bodyweight reduction of its pair fed group. Over the study OXM andliraglutide both significantly reduced body weight, by 10.3% and 8.3%respectively. Measurement of glycemic profile which monitorednon-fasting glucose on days 1, 5, 12, 19, 26 and 29 and fasting glucoseon days 2, 9, 16, 23 and 30 had shown significant improvement of theseparameters, especially for the 6000 nmol/kg (FIG. 9A; FIG. 9B). Oralglucose tolerant test (OGTT) studies were performed on days 2 and day 30(FIG. 10 and FIG. 11, respectively). The results showed that MOD-6031(the amino variant) significantly and dose-dependently improved glucosetolerance with plasma glucose being significantly reduced in the 1000,3000 and 6000 nmoles/kg groups Animals pair-fed to the highest MOD-6031dose exhibited a glucose excursion post glucose dose that was notsignificantly different to controls at any of the time points tested. OnDay 2 of the OGTT studies, the improved glucose profile was associatedwith a delay of the insulin response, which slightly delayed and gavehigher stimulation for AUC 0-120 min (FIG. 10). This can be due toinhibition of gastric emptying induced by MOD-6031's pharmacologicalactivity which results in a delay in glucose release into the blood anda second insulin secretion phase. Day 30 of the OGTT studies wasassociated with a reduced insulin response compared to controls showingthat the compound improved insulin sensitivity (FIG. 11). In addition,MOD-6031 dose-dependently reduced terminal cholesterol; the reductionobserved with the 6000 nmoles/kg dose of MOD-6031 was significantlygreater than that of pair-fed counterparts (FIG. 12). All of thesepharmacological improvements in body weight, food intake, glycemic andlipid profiles were greater not only than animals treated bi-daily withOXM or liraglutide, but they were also significantly greater than theeffects observed in pair-fed counterparts.

Terminal blood level of MOD-6031(PEG-S-MAL-FMS-OXM) and its hydrolyzedcompounds (PEG-S-MAL-FMS and OXM) were measured using an LC-MS/MSqualified method. Results showed dose dependent concentrations for theMOD-6031 treated groups (Table 6). Comparison of this data to compoundlevels on day 2 (following single administration) showed that OXMpeptide were not accumulated during the study period when administeredtwice a week. PEG-S-MAL-FMS and PEG-S-MAL-FMS-OXM showed to moderateaccumulation over the study (Table 6). The actual concentration ofMOD-6031 and OXM peptide for the top dose of MOD-6031 at 24 h post lastinjection (Day 33) were 490 μg/ml and 0.37 μg/ml, respectively. Allsamples from control animals were below the lower limit of the assay.

TABLE 6 Comparison of Plasma Concentrations 24 Hours Following SingleDose (Day 2) and Last Injection of Repeat MOD-6031 Dosing Regimen (Day33). Dose: compound: Day 2 Day 33 Increased by 1000 PEG-S-MAL-FMS-OXM51.57 67.51 1.31 3000 PEG--S-MAL-FMS-OXM 183.33 266.75 1.46 6000PEG--S-MAL-FMS-OXM 296.33 493.60 1.67 1000 OXM 0.07 0.09 1.29 3000 OXM0.23 0.23 1.00 6000 OXM 0.38 0.37 0.97 Dose*: compound: Day 2 Day 33Increased by 1000 PEG--S-MAL-FMS 65.73 78.04 1.19 3000 PEG--S-MAL-FMS211.67 295.75 1.40 6000 PEG--S-MAL-FMS 359.33 740.00 2.06 *Dosesincluding impurities are 1515, 4545, and 9090 nmol/kg

Example 6 Improvement of Pharmacokinetic Parameters by MOD-6031 Variantin Ob/Ob Mouse Model

Results

Three groups (n=12) of ob/ob mice were singly administered with 1000,3000 and 6000 nmol/kg of MOD-6031 and were bled at 4, 8, 24, 36, 48, 72,96 and 120 h post administration (n=3 per time point) for PK analysisand the quantity of MOD-6031 and its compounds concentrations determinedLC-MS/MS method. Pharmacokinetic parameters such as Cmax, Tmax, AUC,T1/2C1 and Vz were calculated for MOD-6031 (PEG-S-MAL-FMS-OXM) and itshydrolyzed products; PEG-S-MAL-FMS-NHS and OXM, these parameters arepresented in Table 7a, 7b and 7c, respectively. AUC 0-∞ was within 15%of AUC 0-t for all components at all doses, indicating that the samplingschedule was adequate to characterize the pharmacokinetic profile ofeach component. For all three components, exposure appeared to bedose-proportional. In general, Cmax and AUC 0-∞ increased with dose andin approximately the same proportion as the increase in dose.

Parameters for each component are expressed in molar concentrations inTable 8. Cmax values were approximately equivalent for PEG-S-MAL-FMS-OXMand PEG-S-MAL-FMS-NHS and lower for OXM. The observed T_(1/2) forPEG-S-MAL-FMS-OXM and OXM were approximately 9 and 12 hours,respectively. The terminal T₁₁₂ for PEG-S-MAL-FMS-NHS was much longer,approximately 30 hours. All samples from control animals and all samplescollected prior to dosing were below the lower limit of the assay.

The pharmacokinetic and pharmacological data confirm the long actingproperties of MOD-6031. Twice a week dose of 3000 nmoles/kg of MOD-6031significantly reduced body weight and food consumption which wascomparable to twice a day of the OXM peptide treatment arm administeredat a 6000 nmoles/kg dose leading also to a significant reduction in drugload.

TABLE 7a PEG-S-MAL-FMS-OXM Pharmacokinetic Parameters Following SCInjection of 1000, 3000, or 6000 nmoles/kg 1000 3000 6000 nmol/kg,nmol/kg, nmol/kg, Parameter Units 34.9 mg/kg 105 mg/kg 210 mg/kg Cmaxμg/mL 70.2 224 311 Tmax hr 8.00 8.00 8.00 AUC_(0-t) hr * μg/mL 1840 633010700 AUC_(0-∞) hr * μg/mL 1850 6330 10700 T_(1/2) hr 8.57 8.80 12.3CL/F mL/hr/kg 18.9 16.5 19.5 Vz/F mL/kg 234 210 346 Cmax/D(μg/mL)/(mg/kg) 2.01 2.14 1.48 AUC_(0-∞)/D (hr * μg/mL)/ 52.9 60.5 51.3(mg/kg)

TABLE 7b PEG-S-MAL-FMS-NHS Pharmacokinetic Parameters Following SCInjection of 1000, 3000, or 6000 nmoles/kg of MOD-6031 1000 3000 6000nmol/kg, nmol/kg, nmol/kg, Parameter Units 34.9 mg/kg 105 mg/kg 210mg/kg Cmax μg/mL 65.7 212 407 Tmax hr 24.0 24.0 36.0 AUC_(0-t) hr *μg/mL 3060 10700 22800 AUC_(0-∞) hr * μg/mL 3280 11200 25800 T_(1/2) hr33.5 22.8 35.0 CL/F mL/hr/kg 14.0 12.4 10.8 Vz/F mL/kg 678 408 544Cmax/D (μg/mL)/(mg/kg) 1.43 1.52 1.46 AUC_(0-∞)/D (hr * μg/mL)/(mg/kg)71.3 80.5 92.8 Note: Due to PEG-S-MAL-FMS-NHS impurity in the dosingsolutions, the administered doses of PEG-S-MAL-FMS-NHS (MOD-6031 plusPEG-S-MAL-FMS-NHS impurity) were 1515, 4545, and 9090 nmol/kg instead of1000, 3000 and 6000 nmol/kg, respectively.

TABLE 7c OXM Pharmacokinetic Parameters Following SC Injection of 1000,3000, or 6000 nmoles/kg of MOD-6031 1000 3000 6000 nmol/kg, nmol/kg,nmol/kg, Parameter Units 34.9 mg/kg 105 mg/kg 210 mg/kg Cmax μg/mL 0.1590.365 0.749 Tmax hr 8.00 8.00 8.00 AUC_(0-t) hr * μg/mL 3.19 9.29 18.5AUC_(0-∞) hr * μg/mL NC 9.42 18.5 T_(1/2) hr NC 11.7 11.8 CL/F mL/hr/kgNC 1420 1440 Vz/F mL/kg NC 23900 24400 Cmax/D (μg/mL)/(mg/kg) 0.03570.0274 0.0280 AUC_(0-∞)/D (hr * μg/mL)/(mg/kg) NC 0.705 0.694 NC = dueto the shape of the concentration versus time profile, parameters couldnot be calculated

TABLE 8 Pharmacokinetic Parameters Comparing the Three Components on aMolar Basis Dose^(a) C_(max)/D AUC_(0-t)/D nmol/ C_(max) (nmol/mL)/AUC_(0-t) (hr * nmol/mL)/ T_(1/2) kg Component nmol/mL (μmol/kg) hr *nmol/mL (μmol/kg) Hr 1000 PEG-S- 2.01 2.01 52.6 52.6 8.57 MAL-FMS- OXM1515 PEG-S- 2.16 1.43 100 66.0 33.5 MAL-FMS- NHS^(a) 1000 OXM 0.03570.0357 0.716 0.716 NC 3000 PEG-S- 6.42 2.14 181 60.3 8.80 MAL-FMS- OXM4545 PEG-S- 6.96 1.53 353 77.7 22.8 MAL-FMS- NHS^(a) 3000 OXM 0.08210.0273 2.09 0.697 11.7 6000 PEG-S- 8.90 1.48 307 51.2 12.3 MAL-FMS- OXM9090 PEG-S- 13.4 1.47 750 82.5 35.0 MAL-FMS- NHS^(a) 6000 OXM 0.1680.0280 4.15 0.692 11.8 ^(a)Doses of PEG-S-MAL-FMS-NHS accounts forimpurities (MOD-6031 plus PEG-S-MAL-FMS-NHS impurity).

MOD-6031 dose-dependently reduced terminal glucose and markedly reducedinsulin in the animals (p<0.01 FIG. 27), indicating that MOD-6031treatment improved insulin sensitivity. For both variables the reductionobserved with the 6000 nmoles/kg dose of MOD-6031 was significantlygreater than that of pair-fed counterparts (p<0.001). Liraglutide had nostatistically significant effect on plasma insulin or glucose at thestudy termination. In contrast, oxyntomodulin significantly reduced bothparameters (p<0.05 for glucose, p<0.001 for insulin).

Example 7 Improvement of Body Weight, Glycemic and Lipid Profiles byPEG30-FMS-OXM Compared to PEG30-Fmoc-OXM and PEG30-EMCS-OXM in Ob/ObMouse Model

The ob/ob mouse model were used as a genetic model of diabetes in thisstudy in order to evaluate the pharmacology efficacy of MOD-6031(PEG30-S-MAL-FMS-OXM) versus its slow rate hydrolysis variant(PEG30-S-MAL-Fmoc-OXM) and its non-reversible form whereN-(epsilon-Maleimidocaproyloxy)succinimide (EMCS) replaces Fmoc aslinker (PEG30-EMCS-OXM). In all those three PEGylated conjugates, thelinker is side directed to the N amino terminal of the OXM peptide.

This study compared the pharmacology efficacy of MOD-6031,PEG30-Fmoc-OXM and PEG30-EMCS-OXM, when administered every four days at6000 nmol/kg, while PEG-SH was used as study control. The measuredpharmacological parameters were body weight, food and water intake,glucose and insulin control and lipid profile. Administration of allthree conjugates significantly reduced body weight and food intakecompared to vehicle (PEG-SH) group during the first two or three weeksof the study (FIG. 13; FIG. 14), while only MOD-6031 exhibit this trenduntil study termination and to a greater extent. Final reduction changesin body weight (on day 33) compared to control (PEG-SH) were 25.4%,5.1%, 2.4% for MOD-6031, PEG30-Fmoc-OXM and PEG30-EMCS-OXM,respectively. Only MOD-6031 displayed significantly lower body weightvalues compared to control. The reduction change in body weight ofPEG30-Fmoc-OXM compared to its pair-fed group was insignificant (2.6%).Body weight reductions were observed following each drug injection forMOD-6031 and PEG30-Fmoc-OXM, while for PEG30-EMCS-OXM, the weightreductions occurred only on days that dosing was followed by anovernight fast. The same profiles have been observed for the reductionin food intake. Measurement of glycemic parameters along the study hadshown significant improvement of non-fasting glucose for MOD-6031 group(FIG. 15A) and significant improvement of fasting glucose for MOD-6031and PEG30-Fmoc-OXM groups (FIG. 15B). OGTT procedures were performed ondays 2 and 30 (FIGS. 16 and 17, respectively). On day 2 OGTT, MOD-6031and PEG30-Fmoc-OXM significantly improved glucose tolerance with plasmaglucose being significantly reduced and insulin secretion significantlyincreased in parallel (FIG. 16). Pair-fed group animals exhibited aglucose excursion post glucose dose that was not significantly differentfrom control at any of the time points tested. On Day 30 OGTT, thesignificant improved glucose profile was observed for both MOD-6031 andPEG30-Fmoc-OXM, however to a lesser extent to the latter. In addition,reduced insulin response compared to controls was observed in bothgroups, suggesting the compounds improved insulin sensitivity (FIG. 17).Terminal plasma samples which were analyzed for lipidic profiles andfructosamine showed significant reduction for both examinations by bothMOD-6031 and PEG30-Fmoc-OXM (FIG. 18; FIG. 19). In both instances, as inall other study results, MOD-6031 exhibited supremacy overPEG30-Fmoc-OXM.

Example 8 In-Vitro Characterization of the Ex-Vivo Hydrolysis Rate ofMOD-6031

This study was conducted in order to characterize and compare theex-vivo hydrolysis rate of MOD-6031 under different conditions:different pH, temperatures, and plasma of different species.

Materials and Methods

A bioanalytical method was validated for the determination ofPEG-S-MAL-FMS-OXM, PEG-S-MAL-FMS-NHS, and OXM in K2EDTA rat and monkeyplasma by liquid chromatography atmospheric pressure ionization tandemmass spectrometry (LC-MS/MS). Stable labelled PEG-S-MAL-FMS-OXM, stablelabelled PEG-S-MAL-FMS-NHS, and ¹³C24, ¹⁵N4-OXM were used as theinternal standards for PEG-S-MAL-FMS-OXM, PEG-S-MAL-FMS and OXM,respectively. PEG-S-MAL-FMS-OXM, PEG-S-MAL-FMS-NHS, and OXM and theirinternal standards were extracted from the tested plasma sample byprotein precipitation extraction at a low pH using acetonitrile. Afterevaporation to dryness and reconstitution, the extracts were analysed byLC-MS/MS. Calibration curves for PEG-S-MAL-FMS-OXM, PEG-S-MAL-FMS-NHSand OXM were prepared freshly for all data sets and were used toquantify the analysed component.

Different pH values were achieved by using phosphate buffer at pH 6.8,7.4 and 7.8. Incubation at temperatures of 35° C., 37° C. and 41° C. wasexamined in rat plasma. Comparison of hydrolysis rates of MOD-6031incubated in rat, cynomolgus monkey or human plasma was evaluated at 37°C. For human plasma, both pooled and individual samples were measuredusing plasma derived from male and female subjects. MOD-6031 (400 μg/mlof total material) was added to tubes containing the relevant plasma orbuffer (N=3), and samples were incubated for 0 (immediately after addingthe material), 4, 8, 24, 48 and 72 h under the above differentconditions. The hydrolysis was stopped at the designated time point byfreezing the sample at −70° C. DPPIV inhibitor (1%) and aprotinin (500KIU/ml) were added to plasma samples prior to the addition of theMOD-6031, in order to avoid unrelated and non-specific cleavage byproteolytic enzymes. For each condition, three independent samples wereprepared. Samples were incubated at a given temperature of either 35°C., 37° C. or 41° C. All samples were stored at −70° C. prior toanalysis. MOD-6031 (PEG-S-MAL-FMS-OXM), OXM and PEG-S-MAL-FMS-NHSconcentrations were quantified utilizing a LC-MS/MS method. MOD-6031hydrolysis profiles were established and hydrolysis rates in differentplasma matrices were calculated.

The explored conditions were:

-   -   a. pH, wherein hydrolysis was tested at pH 6.8, 7.4 and 7.8;    -   b. Temperature, wherein hydrolysis was tested at temperatures of        35° C., 37° C., and 41° C.; and    -   c. Plasma source, wherein hydrolysis was tested in plasma        samples obtained from rat, cynomolgus monkeys and human. For        human plasma, both pooled and individual samples were used, and        hydrolysis rates were measured separately for plasma from males        and females.

MOD-6031 (400 μg/ml of total material) was incubated under the differentconditions for up to 72 h. At designated time points, samples were takenfor LC-MS/MS analysis. MOD-6031, and its degradation products OXM andPEG-S-MAL-FMS-NHS, were quantitated, and pharmacokinetic analysis wasperformed accordingly.

The results indicated that pH level has an effect on MOD-6031 hydrolysisrate; at a higher pH (pH 7.8) the hydrolysis rate was higher compared tothe hydrolysis rate at a lower pH (pH 6.8) (Table 9, FIGS. 20A-20C).With regard to temperature, incubation at 41° C. resulted in a higherhydrolysis rate (Table 10, FIGS. 21A-21C) compared with hydrolysis ratesfor incubation at 35° C. or 37° C. MOD-6031 had a comparable hydrolysisrate in most of the plasma samples as reflected by similar clearancerates and similar increasing of OXM and PEG-S-MAL-FMS-NHS concentrationsin the measured matrices (Table 11, FIGS. 22A-22C). There wasvariability in the clearance rate of OXM in different lots of the samespecies and between the species. PEG-S-MAL-FMS-NHS clearance rate wasvery similar in different plasma species.

Conclusion:

The hydrolysis rates and pattern of hydrolysis of MOD-6031 incubated inplasma from rat, monkey and human matrices were very similar and did notexhibit significant differences more than was observed from differentindividuals per each species.

TABLE 9 PK analysis of PEG-S-MAL-FMS-OXM, OXM and PEG-S-MAL-FMS-NHS indifferent pH Phosphate Phosphate Phosphate Parameter Unit pH = 6.8 pH =7.4 pH = 7.8 PEG-S-MAL-FMS-OXM Tmax h 0 0 0 Cmax μg/ml 283 272 286AUC_(0-t) μg/ml * h 10566 5973 3626 AUC_(0-∞) μg/ml * h 13605 6209 3640T1/2 h 33.7 15.2 8.8 AUC_(0-t/0-∞) 0.78 0.96 1.00 MRT_(0-∞) h 47.4 20.310.7 Vz/F_obs (mg)/(μg/ml) 2.95 2.92 2.89 Cl/F_obs (mg)/(μg/ml)/h 0.0610.133 0.227 OXM Tmax h 72 48 24 Cmax μg/ml 27 30 35.3 AUC_(0-t) μg/ml *h 1279 1759 1979 AUC_(0-∞) μg/ml * h N/a N/a 4772 T1/2 h N/a N/a 82.2AUC_(0-t/0-∞) N/a N/a 0 MRT_(0-∞) h N/a N/a 127 Vz/F_obs (mg)/(μg/ml)N/a N/a 9.9 Cl/F_obs (mg)/(μg/ml)/h N/a N/a 0 PEG-S-MAL-FMS-NHS Tmax h72 72 48 Cmax μg/ml 145 157 161 AUC_(0-t) μg/ml * h 7180 9217 10200AUC_(0-∞) μg/ml * h N/a N/a N/a T1/2 h N/a N/a N/a AUC_(0-t/0-∞) N/a N/aN/a MRT_(0-∞) h N/a N/a N/a Vz/F_obs (mg)/(μg/ml) N/a N/a N/a Cl/F_obs(mg)/(μg/ml)/h N/a N/a N/a

TABLE 10 PK analysis of PEG-S-MAL-FMS-OXM, OXM and PEG- S-MAL- FMS-NHSin different temperatures Parameter Unit Rat 35° C. Rat 37° C. Rat 41°C. PEG-S-MAL-FMS-OXM Tmax h 0 0 0 Cmax μg/ml 319 307 325 AUC_(0-t)μg/ml * h 6843 6397 4603 AUC_(0-∞) μg/ml * h 6867 6412 4617 T1/2 h 8.49.1 6.0 AUC_(0-t/0-∞) 1.00 1.00 1.00 MRT_(0-∞) h 14.7 14.7 9.6 Vz/F_obs(mg)/(μg/ml) 1.45 1.70 1.54 Cl/F_obs (mg)/(μg/ml)/h 0.120 0.129 0.179OXM Tmax h 24 24 24 Cmax μg/ml 13.8 12.3 11.8 AUC_(0-t) μg/ml * h 469414 389 AUC_(0-∞) μg/ml * h 471 416 389 T1/2 h 7.6 7.9 6.2 AUC_(0-t/0-∞)1.00 1.00 1.00 MRT_(0-∞) h 24.6 24.1 18.1 Vz/F_obs (mg)/(μg/ml) 9.3010.92 9.24 Cl/F_obs (mg)/(μg/ml)/h 0.850 0.961 1.028 PEG S-MAL--FMS Tmaxh 48 48 24 Cmax μg/ml 155.3333333 143 150 AUC_(0-t) μg/ml * h 9073 83828800 AUC_(0-∞) μg/ml * h N/a N/a 48287 T1/2 h N/a N/a 213.3AUC_(0-t/0-∞) N/a N/a 0.18 MRT_(0-∞) h N/a N/a 317.7 Vz/F_obs(mg)/(μg/ml) N/a N/a 2.55 Cl/F_obs (mg)/(μg/ml)/h N/a N/a 0.008

TABLE 11 PK values for OXM, PEG-S-MAL-FMS and PEG-S-MAL-FMS-OXM MonkeyMonkey (lot# (lot# Human CYN1 CYN12 male Human Human Parameter Unit Rat28423) 8421) (pool) male A male B OXM Tmax h 24 48 4 8 4 24 Cmax μg/ml12.3 21.3 5.0 6.2 4.3 18.3 AUC_(0-t) μg/ml * h 414 1211 67 207 91 1232AUC_(0-∞) μg/ml * h 416 N/a 67 212 92 1788 T_(1/2) h 8 N/a 9 12 11 51AUC_(0-t/0-∞) 0.995 N/a 0.998 0.977 0.997 0.689 MRT_(0-∞) h 24 N/a 9 2315 83 Vz/F_obs (mg)/(μg/ml) 10.92 N/a 78.61 32.42 68.54 16.53 Cl/F_obs(mg)/(μg/ml)/h 0.961 N/a 6.000 1.883 4.361 0.224 PEG-S-MAL-FMS-NHS Tmaxh 48 48 24 48 48 24 Cmax μg/ml 143.0 190.3 136.5 164.7 128.0 138AUC_(0-t) μg/ml * h 8382 10496 11443 9332 8023 8261 AUC_(0-∞) μg/ml * hMissing N/a 69979 N/a N/a 32732 T_(1/2) h Missing N/a 342 N/a N/a 153AUC_(0-t/0-∞) Missing N/a 0.164 N/a N/a 0.252 MRT_(0-∞) h Missing N/a502 N/a N/a 228 Vz/F_obs (mg)/(μg/ml) Missing N/a 2.82 N/a N/a 2.69Cl/F_obs (mg)/(μg/ml)/h Missing N/a 0.006 N/a N/a 0.012PEG-S-MAL-FMS-OXM Tmax h 0 0 0 0 0 0 Cmax μg/ml 306.7 344.0 295.5 258.3272.0 267 AUC_(0-t) μg/ml * h 5971 7776 2284 5224 3003 2694 AUC_(0-∞)μg/ml * h 5983 7793 2321 5232 3011 2698 T_(1/2) h 7.6 8.8 4.1 7.1 5.65.2 AUC_(0-t/0-∞) 1.00 1.00 0.98 1.00 1.00 1.00 MRT_(0-∞) h 13 15 5 14 87 Vz/F_obs (mg)/(μg/ml) 0.62 0.54 1.02 0.66 1.07 1.11 Cl/F_obs(mg)/(μg/ml)/h 0.056 0.043 0.172 0.064 0.133 0.148 Human Human HumanHuman Human female female female female Parameter Unit male C (pool) A BC OXM Tmax h 8 24 8 4 8 Cmax μg/ml 7.3 32.1 4.7 4.1 6.5 AUC_(0-t)μg/ml * h 168 1780 98 78 141 AUC_(0-∞) μg/ml * h 169 4347 99 78 142T_(1/2) h 11 83 11 11 11 AUC_(0-t/0-∞) 0.997 0.409 0.996 0.997 0.996MRT_(0-∞) h 17 128 16 14 17 Vz/F_obs (mg)/(μg/ml) 36.27 11.00 64.4479.44 46.34 Cl/F_obs (mg)/(μg/ml)/h 2.370 0.092 4.050 5.116 2.823PEG-S-MAL-FMS-NHS Tmax h 24 24 24 24 48 Cmax μg/ml 136 161.7 127.0 130127.0 AUC_(0-t) μg/ml * h 8244 9997 8119 7842 7706 AUC_(0-∞) μg/ml * h35933 124011 128128 66324 N/a T_(1/2) h 171 521 687 344 N/aAUC_(0-t/0-∞) 0.229 0.081 0.063 0.118 N/a MRT_(0-∞) h 255 760 999 505N/a Vz/F_obs (mg)/(μg/ml) 2.75 2.42 3.10 2.99 N/a Cl/F_obs(mg)/(μg/ml)/h 0.011 0.003 0.003 0.006 N/a PEG-S-MAL-FMS-OXM Tmax h 0 00 0 0 Cmax μg/ml 267 255.3 283.0 237 257.0 AUC_(0-t) μg/ml * h 2737 32583184 2924 2783 AUC_(0-∞) μg/ml * h 2741 3266 3193 2936 2789 T_(1/2) h5.2 5.6 5.8 6.1 5.4 AUC_(0-t/0-∞) 1.00 1.00 1.00 1.00 1.00 MRT_(0-∞) h 79 8 9 7 Vz/F_obs (mg)/(μg/ml) 1.10 0.83 1.04 1.19 1.12 Cl/F_obs(mg)/(μg/ml)/h 0.146 0.102 0.125 0.136 0.143

Example 9 In-Vitro Evaluation of Protection Provided by the PEG Moietyof MOD-6031 from Dipeptidyl Peptidase IV (DPPIV) Digestion

MOD-6031, OXM peptide and PEG-EMCS-OXM were incubated with DPPIV anddigestion of each was tested by RP-HPLC. The digested and non-digestedforms were identified and measured.

First, a preliminary examination of OXM peptide degradation at twodifferent pH levels (pH=6 and pH=7) in 10 mM Tris buffer was evaluated.Each reaction was incubated at 37° C. for 1 hour. After the incubation,50 μl of the reaction was diluted with 100 μl of 0.1% TFA in DDW. 10 μlof this solution was then loaded on a RP-HPLC Intrada WP-RP 2×50 mm, 3μm, 300 Å column (a total of 3.3 μg).

The non-digested and the digested forms of OXM and MOD-6031 wereidentified using RP-HPLC column. The elution time of the cleaved, inactive form of OXM, OXM 3-37, differs from OXM peptide by 0.2 minPercentage digestion was evaluated by measuring percentage relativearea. For each reaction, a control sample without DPPIV was prepared andmeasured.

MOD-6031 and PEG-EMCS-OXM were incubated with DPPIV and percentagedigestion was measured. The reactions conditions were the same asdescribed for the OXM peptide above.

The enzyme dipeptidyl peptidase IV (DPPIV) is an intrinsic membraneglycoprotein, expressed in most cell types and cleaves dipeptides fromthe N-terminus of polypeptides. OXM digestion by DPPIV has beendemonstrated in vitro and in vivo, and is to considered as the maincause for the short half-life of the peptide in the bloodstream. OXM iscleaved between amino acids at positions 2 and 3, resulting in thenon-active form OXM 3-37. In this study the digestion by DPPIV of OXMpeptide linked to PEG in the reversible and non-reversible conjugations,MOD-6031 and PEG-EMCS-OXM, respectively, was examined.

Preliminary evaluation of OXM peptide degradation rate by DPPIV enzymeat pH=6 vs pH=7, indicated that at pH=6 DPPIV enzyme was more effective,with % relative area of 46.12 for OXM 3-37 at pH=6, compared to 26.52 atpH=7 (FIGS. 23-24, Tables 12-15) after 1 hour incubation at 37° C.Therefore the digestion study of MOD-6031 was performed at pH=6, whichis also a preferable condition for MOD-6031 hydrolysis prevention.

Percent digestion of MOD-6031 and PEG-EMCS-OXM by DPPIV were measured.Degradation of the MOD-6031 conjugate was evaluated following incubationwith DPPIV (FIG. 25, Table 17) and analysis by RP-HPLC column using thesame conditions that were used for the OXM peptide. As a negativecontrol, reactions with varied pH and/or temperature but without addedDPPIV were run in order to confirm that OXM is not hydrolyzed. As apositive control, reactions evaluating hydrolysis of OXM that was not apart of a conjugate was measured. (FIG. 25, Table 16).

No degradation of MOD-6031 was observed following incubation of MOD-6031in the absence of enzyme DPPIV, and therefore, no hydrolysis of OXM, Thepercentage of relative area was 98.28. Two reactions with DPPIV 1×[DPPIVconcentration](Table 17) and 10×[DPPIV concentration (Table 18) wereperformed In both reactions no degradation of OXM was observed and thepercentage relative area of MOD-6031 were 98.49 and 98.24, respectively.

The non-reversible PEGylated PEG-EMCS-OXM was also tested for OXMdegradation by DPPIV in the same manner (FIG. 26, Table 20). As acontrol, a reaction without DPPIV was prepared (FIG. 26, Table 19). Inboth reactions, no degradation of the conjugates was observed. Thepercentage relative area of PEG-EMCS-OXM was 98.48 and 99.09,respectively.

Based on the results presented here, it can be concluded that OXMconjugated to a PEG moiety via a hydrolysable or a non-hydrolysablelinker is protected from degradation by DPPIV.

TABLE 12 Degradation assays of OXM at pH = 6 Peak Retention AreaRelative No. Name Time min mAU * min Area % 1 6.823 0.177 0.44 2 OXM7.227 38.996 96.62 3 7.417 0.924 2.29 4 8.340 0.265 0.66 Total: 40.362100.00

TABLE 13 Degradation assays of OXM + DPPIV at pH = 6 Retention AreaRelative No. Peak Name Time min mAU * min Area % 1 7.023 0.276 0.79 2OXM 7.227 18.121 51.69 3 OXM 3-37 7.417 16.165 46.12 4 7.610 0.314 0.905 7.767 0.178 0.51 Total: 35.054 100.00

TABLE 14 Degradation assays of OXM at pH = 7 Peak Retention Time Area %Relative No. Name min mAU * min Area 1 6.820 0.201 0.39 2 OXM 7.22349.397 96.65 3 7.417 1.266 2.48 4 8.340 0.245 0.48 Total: 51.109 100.00

TABLE 15 Degradation assays of OXM + DPPIV at pH = 7 Retention Area %Relative No. Peak Name Time min mAU*min Area 1 6.823 0.166 0.30 2 OXM7.223 39.441 72.28 3 OXM 3-37 7.417 14.469 26.52 4 7.610 0.318 0.58 57.770 0.174 0.32 Total: 54.568 100.00

TABLE 16 Degradation assays of MOD-6031 at pH = 6 Peak Retention AreaRelative No. Name Time min mAU * min Area % 1 7.240 0.284 0.56 2 8.2800.234 0.46 3 9.830 0.045 0.09 4 MOD- 17.930 49.565 98.28 6031 5 18.9170.029 0.06 6 19.203 0.036 0.07 7 19.403 0.240 0.48 50.433 100.00

TABLE 17 Degradation assays of MOD-6031 + DPPIV (1X DPPIVconcentration)at pH = 6 Peak Retention Area Relative No. Name Time minmAU * min Area % 1 7.250 0.081 0.17 2 8.310 0.267 0.56 3 9.847 0.0530.11 4 MOD- 17.937 46.994 98.49 6031 5 18.540 0.037 0.08 6 19.190 0.0340.07 7 19.403 0.251 0.53 47.717 100.00

TABLE 18 Degradation assays of MOD-6031 + DPPIV (10X DPPIVconcentration) at pH = 6 MOD- 6031 + DPP4x10 Retention Area Relative No.Peak Name Time min mAU * min Area % 1 7.377 0.061 0.13 2 7.483 0.0040.01 3 8.313 0.315 0.64 4 9.847 0.049 0.10 5 MOD-6031 17.940 48.03698.24 6 18.517 0.104 0.21 7 19.213 0.034 0.07 8 19.410 0.293 0.60 48.896100.00

TABLE 19 Degradation assays of PEG-EMCS-OXM at pH = 6 Retention AreaRelative Area No. Peak Name Time min mAU * min % 1 8.317 0.238 0.56 29.860 0.031 0.07 3 17.547 0.306 0.72 4 PEG-EMCS- 17.847 41.557 98.48 OXM5 18.857 0.046 0.11 6 19.100 0.022 0.05 Total: 42.200 100.00

TABLE 20 Degradation assays of PEG-EMCS-OXM + DPPIV iat pH = 6 RetentionArea Relative Area No. Peak Name Time min mAU * min % 1 8.317 0.280 0.692 10.020 0.024 0.06 3 PEG-EMCS- 17.850 39.952 99.09 OXM 4 18.827 0.0410.10 5 19.140 0.022 0.06 Total: 40.318 100.00

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. A method for the preparation of an oxyntomodulin(OXM) conjugate represented by the structure of formula II:

wherein R₂ is H or SO₃H; said method comprises: reacting MAL-Fmoc-NHS(R₂═H) represented by the following structure:

MAL-FMS-NHS (R₂═SO₃H) represented by the following structure:

with a resin-bound oxyntomodulin of SEQ ID NO:1, wherein the amino sidechains of Lys¹² and Lys³⁰ of said resin-bound oxyntomodulin are eachprotected by a protecting group, to obtain a resin-boundMAL-Fmoc-protected OXM or a resin-bound MAL-FMS-protected OXM,respectively; followed by (b) reaction of the resin-boundMAL-Fmoc-protected OXM or the resin-bound MAL-FMS-protected OXM withsulfhydryl PEG polymer (PEG-SH) and subsequently removing the protectinggroups and said resin; or (c) removal of the protecting group and theresin to provide MAL-Fmoc-OXM and a MAL-FMS-OXM, respectively, andsubsequent reaction of the MAL-Fmoc-OXM or the MAL-FMS-OXM withsulfhydryl PEG polymer (PEG-SH); to yield the structure of formula II,wherein the amino residue of His¹ of said OXM is linked to said Fmoc orFMS.
 2. The method of claim 1, wherein said MAL-FMS-NHS is prepared bymixing MAL-Fmoc-NHS with trifluoroacetic acid and chlorosulfonic acid.3. The method of claim 2, wherein MAL-Fmoc-NHS is dissolved in neattrifluoroacetic acid and 6 equivalents of chlorosulfonic acid dissolvedin neat trifluoroacetic acid is added to the reaction mixture.
 4. Themethod of claim 1, wherein said protecting group of said Lys¹² and Lys³⁰is 1-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidine)ethyl]), (ivDde).
 5. Themethod of claim 1 wherein said reaction with said sulfhydryl PEG polymer(PEG-SH) is conducted under a buffer condition of pH between 6 and 6.5.6. The method of claim 1, wherein said PEG has a molecular weight in therange of 20,000 Da to 40,000 Da.
 7. The method of claim 6, wherein saidPEG has a molecular weight with an average molecular weight of 30,000Da.